Adenovirus vectors and methods for using adenovirus vectors

ABSTRACT

This document provides adenovirus vectors and methods and materials related to using adenovirus vectors. For example, adenoviruses for delivering nucleic acid encoding one or more immunogens (e.g., one or more immunogens associated with a pathogen causing an infection) to cells within a mammal such that the mammal produces an effective immune response against the immunogen(s) are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/996,740, filed Aug. 18, 2020, which also claims the benefit of U.S.Provisional Application Ser. No. 63/066,740, filed on Aug. 17, 2020, andthis application is a continuation-in-part of International PCT PatentApplication Serial No. PCT/US2021/046333, filed Aug. 17, 2021, whichalso claims the benefit of U.S. Provisional Application Ser. No.63/066,740, filed on Aug. 17, 2020. The disclosures of the priorapplications are considered part of (and is incorporated by referencein) the disclosure of this application.

SEQUENCE LISTING

This document includes a sequence listing submitted to the United StatesPatent and Trademark Office via the electronic filing system as an ASCIItext file. The sequence listing, which is incorporated-by-referenceherein, is titled “07039_1964002_ST25.txt,” was created on Aug. 6, 2021,and has a size of 823 kilobytes.

TECHNICAL FIELD

This document relates to adenovirus vectors and methods and materialsrelated to using adenovirus vectors. For example, adenovirus vectors canbe used to deliver one or more immunogens (e.g., one or more immunogensassociated with a pathogen causing an infection) to cells within amammal such that the mammal produces an effective immune responseagainst the immunogen(s).

BACKGROUND INFORMATION

An infectious disease caused by a coronavirus known as COVID-19 wasfirst reported to the World Health Organization (WHO) Country Office inChina on Dec. 31, 2019. As of Jun. 3, 2020, approximately 6,287,771confirmed cases of COVID-19, including 379,941 deaths, have beenreported to the WHO (covid19.who.int/).

SUMMARY

This document relates to adenovirus vectors and methods and materialsrelated to using adenovirus vectors. For example, this document providesadenovirus vectors, nucleic acid molecules encoding adenovirus vectors,cell lines containing adenovirus vectors, and methods for usingadenovirus vectors to deliver nucleic acid to cells in vitro or in vivo.This document also provides methods and materials for using adenovirusvectors to induce immune responses within a mammal (e.g., a human). Insome cases, adenovirus vectors (e.g., single-cycle adenovirus (SC-Ad)vectors) can be used to deliver one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, or more) immunogens thattrigger an immune response within a mammal (e.g., a human).

As demonstrated herein, SC-Ads can be engineered to express one or moreimmunogens as fused and secreted polypeptides that can induce aneffective immune response against those immunogens. For example,administration of SC-Ads expressing C. difficile TcdA/B fusionpolypeptides protected mice and Syrian Hamsters from lethal toxinchallenge for extended periods (e.g., over 36 weeks) after a singleimmunization.

Having the ability to produce immune responses effectively against viraland/or bacterial pathogens (e.g., a coronavirus) in mammals (e.g.,humans) can improve survival and minimize the impact of the infection.Adenovirus vectors encoding one or more immunogens can be used toprovide a mammal with sustained, long-term immunity against aninfectious pathogen (e.g., a coronavirus). For example, adenovirusvectors encoding one or more immunogens associated with COVID-19 (e.g.,one or more immunogens derived from SARS-CoV-2) can be used as a robustvaccine in the COVID-19 pandemic to generate humoral immunity againstboth primary infections and recurrences.

In general, one aspect of this document features a SC-Ad, where theSC-Ad has a genome lacking at least a portion of a nucleic acid sequencethat encodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes a nucleic acidsequence encoding a coronavirus immunogen. The adenovirus polypeptidecan be a fiber polypeptide, a V polypeptide, a hexon polypeptide, apenton base polypeptide, or a pIIIa polypeptide. The coronavirusimmunogen can include a coronavirus Spike polypeptide or an immunogenicfragment thereof. The coronavirus immunogen can consist of or canconsist essentially of an amino acid sequence set forth in any one ofSEQ ID NOs:1-4. The SC-Ad also can include a nucleic acid sequenceencoding an adjuvant polypeptide. The adjuvant polypeptide can be agranulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide,an interleukin 4 (IL-4) polypeptide, an interleukin 21 (IL-21)polypeptide, a CD40 ligand (CD40L) polypeptide, a 4-1BB ligand (4-1BBL)polypeptide, a transforming growth factor beta (TGF-β) polypeptide, aClostridium difficile TcdA polypeptide, a C. difficile TcdB polypeptide,or a biologically active fragment thereof. The coronavirus Spikepolypeptide can be fused to the adjuvant polypeptide. The coronavirusSpike polypeptide fused to the adjuvant polypeptide can consistessentially of or can consist of an amino acid sequence set forth in SEQID NO:5. The SC-Ad also can include a nucleic acid sequence encoding achaff polypeptide. The chaff polypeptide can be a fragment of an ACE2polypeptide. The fragment of an ACE2 polypeptide can include theextracellular region of an ACE2 polypeptide and can lack a transmembranedomain. The chaff polypeptide can consist essentially of or can consistof an amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9. Thecoronavirus Spike polypeptide can be fused to the chaff polypeptide.

In another aspect, this document features a composition including aSC-Ad, where the SC-Ad has a genome lacking at least a portion of anucleic acid sequence that encodes an adenovirus polypeptide, where theSC-Ad includes the adenovirus polypeptide, and where the SC-Ad includesa nucleic acid sequence encoding a coronavirus immunogen.

In another aspect, this document features methods for inducing an immuneresponse against a coronavirus in a mammal. The methods can include, orconsist essentially of, administering to a mammal i) a SC-Ad, where theSC-Ad has a genome lacking at least a portion of a nucleic acid sequencethat encodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes a nucleic acidsequence encoding a coronavirus immunogen; or ii) a compositionincluding a SC-Ad, where the SC-Ad has a genome lacking at least aportion of a nucleic acid sequence that encodes an adenoviruspolypeptide, where the SC-Ad includes the adenovirus polypeptide, andwhere the SC-Ad includes a nucleic acid sequence encoding a coronavirusimmunogen, under conditions where the SC-Ad infects a cell of themammal, and where expression of the immunogen in the cell leads toinduction of the immune response. The mammal can be a human. Thecoronavirus can be a betacoronavirus. The betacoronavirus can beSARS-CoV-2. The administering can include mucosal delivery of the SC-Ad.

In another aspect, this document features a SC-Ad, where the SC-Ad has agenome lacking at least a portion of a nucleic acid sequence thatencodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes (a) a nucleic acidsequence encoding an immunogen and (b) a nucleic acid sequence encodingan adjuvant polypeptide. The adenovirus polypeptide can be a fiberpolypeptide, a V polypeptide, a hexon polypeptide, a penton basepolypeptide, or a pIIIa polypeptide. The immunogen can be a coronavirusimmunogen. The coronavirus immunogen can include a coronavirus Spikepolypeptide or an immunogenic fragment thereof. The coronavirusimmunogen can consist of or can consist essentially of an amino acidsequence set forth in any one of SEQ ID NOs:1-4. The adjuvantpolypeptide can be a GM-CSF polypeptide, an IL-4 polypeptide, an IL-21polypeptide, a CD40L polypeptide, a 4-1BBL polypeptide, a TGF-βpolypeptide, a Clostridium difficile TcdA polypeptide, a C. difficileTcdB polypeptide, or a biologically active fragment thereof. Thecoronavirus Spike polypeptide can be fused to the adjuvant polypeptide.The coronavirus Spike polypeptide fused to the adjuvant polypeptide canconsist essentially of or can consist of an amino acid sequence setforth in SEQ ID NO:5. The SC-Ad also can include a nucleic acid sequenceencoding a chaff polypeptide. The chaff polypeptide can be a fragment ofan ACE2 polypeptide. The fragment of an ACE2 polypeptide can include theextracellular region of an ACE2 polypeptide and can lack a transmembranedomain. The chaff polypeptide can consist essentially of or can consistof an amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9.

In another aspect, this document features a composition including aSC-Ad, where the SC-Ad has a genome lacking at least a portion of anucleic acid sequence that encodes an adenovirus polypeptide, where theSC-Ad includes the adenovirus polypeptide, and where the SC-Ad includes(a) a nucleic acid sequence encoding an immunogen and (b) a nucleic acidsequence encoding an adjuvant polypeptide.

In another aspect, this document features methods for inducing an immuneresponse against a virus in a mammal. The methods can include, orconsist essentially of, administering to a mammal i) a SC-Ad, where theSC-Ad has a genome lacking at least a portion of a nucleic acid sequencethat encodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes (a) a nucleic acidsequence encoding an immunogen and (b) a nucleic acid sequence encodingan adjuvant polypeptide; or ii) a composition that includes a SC-Ad,where the SC-Ad has a genome lacking at least a portion of a nucleicacid sequence that encodes an adenovirus polypeptide, where the SC-Adincludes the adenovirus polypeptide, and where the SC-Ad includes (a) anucleic acid sequence encoding an immunogen and (b) a nucleic acidsequence encoding an adjuvant polypeptide, under conditions where theSC-Ad infects a cell of the mammal, and where expression of theimmunogen in the cell leads to induction of the immune response. Themammal can be a human. The virus can be a coronavirus, and the immunogencan be associated with the coronavirus. The coronavirus can be abetacoronavirus. The betacoronavirus can be SARS-CoV-2. Theadministering can include mucosal delivery of the SC-Ad.

In another aspect, this document features a SC-Ad, where the SC-Ad has agenome which lacks at least a portion of a nucleic acid sequence thatencodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes (a) a nucleic acidsequence encoding an immunogen and (b) a nucleic acid sequence encodinga chaff polypeptide. The adenovirus polypeptide can be a fiberpolypeptide, a V polypeptide, a hexon polypeptide, a penton basepolypeptide, or a pIIIa polypeptide. The immunogen can be a coronavirusimmunogen. The coronavirus immunogen can include a coronavirus Spikepolypeptide or an immunogenic fragment thereof. The coronavirusimmunogen can consist of or can consist essentially of an amino acidsequence set forth in any one of SEQ ID NOs:1-4. The chaff polypeptidecan be a fragment of an ACE2 polypeptide. The fragment of an ACE2polypeptide can include the extracellular region of an ACE2 polypeptideand can lack a transmembrane domain. The chaff polypeptide can consistessentially of or can consist of an amino acid sequence set forth in SEQID NO:8 or SEQ ID NO:9. The coronavirus Spike polypeptide can be fusedto the chaff polypeptide. The SC-Ad also can include a nucleic acidsequence encoding an adjuvant polypeptide. The adjuvant polypeptide canbe a GM-CSF polypeptide, an IL-4 polypeptide, an IL-21 polypeptide, aCD40L polypeptide, a 4-1BBL polypeptide, a TGF-β polypeptide, aClostridium difficile TcdA polypeptide, a C. difficile TcdB polypeptide,or a biologically active fragment thereof. In another aspect, thisdocument features for a composition including a SC-Ad, where the SC-Adhas a genome which lacks at least a portion of a nucleic acid sequencethat encodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes (a) a nucleic acidsequence encoding an immunogen and (b) a nucleic acid sequence encodinga chaff polypeptide.

In another aspect, this document features methods for inducing an immuneresponse against a virus in a mammal. The methods can include, orconsist essentially of, administering to a mammal i) a SC-Ad, where theSC-Ad has a genome which lacks at least a portion of a nucleic acidsequence that encodes an adenovirus polypeptide, where the SC-Adincludes the adenovirus polypeptide, and where the SC-Ad includes (a) anucleic acid sequence encoding an immunogen and (b) a nucleic acidsequence encoding a chaff polypeptide, or ii) a composition including aSC-Ad, where the SC-Ad has a genome which lacks at least a portion of anucleic acid sequence that encodes an adenovirus polypeptide, where theSC-Ad includes the adenovirus polypeptide, and where the SC-Ad includes(a) a nucleic acid sequence encoding an immunogen and (b) a nucleic acidsequence encoding a chaff polypeptide, under conditions where the SC-Adinfects a cell of the mammal, and where expression of the immunogen inthe cell leads to induction of the immune response. The mammal can be ahuman. The virus can be a coronavirus, and the immunogen can beassociated with the coronavirus. The coronavirus can be abetacoronavirus. The betacoronavirus can be SARS-CoV-2. Theadministering can include mucosal delivery of the SC-Ad.

In another aspect, this document features for a SC-Ad, where the SC-Adhas a genome which lacks at least a portion of a nucleic acid sequencethat encodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes (a) a nucleic acidsequence encoding an immunogen, (b) a nucleic acid sequence encoding anadjuvant polypeptide, and (c) a nucleic acid sequence encoding a chaffpolypeptide. The adenovirus polypeptide can be a fiber polypeptide, a Vpolypeptide, a hexon polypeptide, a penton base polypeptide, or a pIIIapolypeptide. The immunogen can be a coronavirus immunogen. Thecoronavirus immunogen can include a coronavirus Spike polypeptide or animmunogenic fragment thereof. The coronavirus immunogen can consist ofor can consist essentially of an amino acid sequence set forth in anyone of SEQ ID NOs:1-4. The adjuvant polypeptide can be a GM-CSFpolypeptide, an IL-4 polypeptide, an IL-21 polypeptide, a CD40Lpolypeptide, a 4-1BBL polypeptide, a TGF-β polypeptide, a Clostridiumdifficile TcdA polypeptide, a C. difficile TcdB polypeptide, or abiologically active fragment thereof. The chaff polypeptide can be afragment of an ACE2 polypeptide. The fragment of an ACE2 polypeptide caninclude the extracellular region of an ACE2 polypeptide and can lack atransmembrane domain. The chaff polypeptide can consist essentially ofor can consist of an amino acid sequence set forth in SEQ ID NO:8 or SEQID NO:9.

In another aspect, this document features for a composition including aSC-Ad, where the SC-Ad has a genome which lacks at least a portion of anucleic acid sequence that encodes an adenovirus polypeptide, where theSC-Ad includes the adenovirus polypeptide, and where the SC-Ad includes(a) a nucleic acid sequence encoding an immunogen, (b) a nucleic acidsequence encoding an adjuvant polypeptide, and (c) a nucleic acidsequence encoding a chaff polypeptide.

In another aspect, this document features methods for inducing an immuneresponse against a virus in a mammal. The methods can include, orconsist essentially of, administering to a mammal i) a SC-Ad, where theSC-Ad has a genome which lacks at least a portion of a nucleic acidsequence that encodes an adenovirus polypeptide, where the SC-Adincludes the adenovirus polypeptide, and where the SC-Ad includes (a) anucleic acid sequence encoding an immunogen, (b) a nucleic acid sequenceencoding an adjuvant polypeptide, and (c) a nucleic acid sequenceencoding a chaff polypeptide, or ii) a composition including a SC-Ad,where the SC-Ad has a genome which lacks at least a portion of a nucleicacid sequence that encodes an adenovirus polypeptide, where the SC-Adincludes the adenovirus polypeptide, and where the SC-Ad includes (a) anucleic acid sequence encoding an immunogen, (b) a nucleic acid sequenceencoding an adjuvant polypeptide, and (c) a nucleic acid sequenceencoding a chaff polypeptide, under conditions where the SC-Ad infects acell of the mammal, and where expression of the immunogen in the cellleads to induction of the immune response. The mammal can be a human.The virus can be a coronavirus, and the immunogen can be associated withthe coronavirus. The coronavirus can be a betacoronavirus. Thebetacoronavirus can be SARS-CoV-2. The administering can include mucosaldelivery of the SC-Ad.

In another aspect, this document features for a SC-Ad, where the SC-Adhas a genome lacking at least a portion of a nucleic acid sequence thatencodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes a nucleic acidsequence encoding an immunogen expressed by or shed by an allergen. Theadenovirus polypeptide can be a fiber polypeptide, a V polypeptide, ahexon polypeptide, a penton base polypeptide, or a pIIIa polypeptide.The SC-Ad also can include a nucleic acid sequence encoding an adjuvantpolypeptide. The adjuvant polypeptide can be a GM-CSF polypeptide, anIL-4 polypeptide, an IL-21 polypeptide, a CD40L polypeptide, a 4-1BBLpolypeptide, a TGF-β polypeptide, a Clostridium difficile TcdApolypeptide, a C. difficile TcdB polypeptide, or a biologically activefragment thereof. The SC-Ad also can include a nucleic acid sequenceencoding a chaff polypeptide. The chaff polypeptide can be a fragment ofan ACE2 polypeptide. The fragment of an ACE2 polypeptide can include theextracellular region of an ACE2 polypeptide and can lack a transmembranedomain. The chaff polypeptide can consists essentially of or consists ofan amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9.

In another aspect, this document features for a composition including aSC-Ad, where the SC-Ad has a genome lacking at least a portion of anucleic acid sequence that encodes an adenovirus polypeptide, where theSC-Ad includes the adenovirus polypeptide, and where the SC-Ad includesa nucleic acid sequence encoding an immunogen expressed by or shed by anallergen.

In another aspect, this document features methods for inducing an immuneresponse against an allergen in a mammal. The methods can include, orconsist essentially of, administering to a mammal i) a SC-Ad, where theSC-Ad has a genome lacking at least a portion of a nucleic acid sequencethat encodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes a nucleic acidsequence encoding an immunogen expressed by or shed by an allergen, orii) a composition including a SC-Ad, where the SC-Ad has a genomelacking at least a portion of a nucleic acid sequence that encodes anadenovirus polypeptide, where the SC-Ad includes the adenoviruspolypeptide, and where the SC-Ad includes a nucleic acid sequenceencoding an immunogen expressed by or shed by an allergen, underconditions where the SC-Ad infects a cell of the mammal, and whereexpression of the immunogen in the cell leads to induction of the immuneresponse. The mammal can be a human.

In another aspect, this document features for a SC-Ad, where the SC-Adhas a genome lacking at least a portion of a nucleic acid sequence thatencodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes a nucleic acidsequence encoding an immunogen expressed by a cancer cell. Theadenovirus polypeptide can be a fiber polypeptide, a V polypeptide, ahexon polypeptide, a penton base polypeptide, or a pIIIa polypeptide.The SC-Ad also can include a nucleic acid sequence encoding an adjuvantpolypeptide. The adjuvant polypeptide can be a GM-CSF polypeptide, anIL-4 polypeptide, an IL-21 polypeptide, a CD40L polypeptide, a 4-1BBLpolypeptide, a TGF-β polypeptide, a Clostridium difficile TcdApolypeptide, a C. difficile TcdB polypeptide, or a biologically activefragment thereof. The SC-Ad also can include a nucleic acid sequenceencoding a chaff polypeptide. The chaff polypeptide can be a fragment ofan ACE2 polypeptide. The fragment of an ACE2 polypeptide can include theextracellular region of an ACE2 polypeptide and can lack a transmembranedomain. The chaff polypeptide can consist essentially of or can consistof an amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9.

In another aspect, this document features for a composition including aSC-Ad, where the SC-Ad has a genome lacking at least a portion of anucleic acid sequence that encodes an adenovirus polypeptide, where theSC-Ad includes the adenovirus polypeptide, and where the SC-Ad includesa nucleic acid sequence encoding an immunogen expressed by a cancercell.

In another aspect, this document features methods for inducing an immuneresponse against a cancer cell in a mammal. The methods can include, orconsist essentially of, administering to a mammal i) a SC-Ad, where theSC-Ad has a genome lacking at least a portion of a nucleic acid sequencethat encodes an adenovirus polypeptide, where the SC-Ad includes theadenovirus polypeptide, and where the SC-Ad includes a nucleic acidsequence encoding an immunogen expressed by a cancer cell, or ii) acomposition including a SC-Ad, where the SC-Ad has a genome lacking atleast a portion of a nucleic acid sequence that encodes an adenoviruspolypeptide, where the SC-Ad includes the adenovirus polypeptide, andwhere the SC-Ad includes a nucleic acid sequence encoding an immunogenexpressed by a cancer cell, under conditions where the SC-Ad infects acell of the mammal, and where expression of the immunogen in the cellleads to induction of the immune response. The mammal can be a human.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Schematic of an exemplary TcdA/B fusion protein and anexemplary SC-Ad6 plasmid expressing a fusion protein according to someembodiments. (FIG. 1A) A TcdA/B fusion protein includes a human alpha1-antitrypsin (AAT) secretory leader sequence and the RBDs of TcdA andTcdB that are separated by two furin cleavage sites. (FIG. 1B) A SC-Ad6plasmid expressing the TcdA/B fusion using the CMV promoter.

FIGS. 2A-2B. Single-cycle adenovirus expression of C. difficile toxins Aand B fusion protein. Western blot detecting expression of (FIG. 2A)TcdA and (FIG. 2B) TcdB fragments in the cell lysates and media takenfrom uninfected cells, cells infected with an Ad control(SC-Ad6-GFP-luciferase (GL)), and cells infected with SC-Ad6-TcdA/B.

FIGS. 3A-3C. Serum antibody responses in immunized CD1 mice and toxin Achallenge. Male and female CD1 mice (n=10) were vaccinatedintramuscularly (i.m.) with 1×10¹⁰ virus particles of SC-Ad6-PEB1,SC-Ad6-TcdA/B, or PBS. Serum collected at week 6 was assayed by ELISAand a neutralization assay. (FIG. 3A) Serum endpoint titers at week 6were significantly (p=0.0066) higher in female mice immunized withSC-Ad-TcdA/B than in males (Geometric mean with 95% CI). (FIG. 3B)Neutralizing titers for Toxin A were significantly higher in the femaleand male groups compared to sex matched controls (Adjusted p=0.0001 and0.0238 for females and males by Dunn's). (FIG. 3C) Combined male andfemale toxin A survival curve shows significant survival compared to PBSor PEB1 control animals (p<0.0001).

FIGS. 4A-4D. SC-Ad6-TcdA/B provides protection against lethal challengelong after a single immunization. Female CD1 mice (n=5) were vaccinatedi.m. with 1×10¹⁰ virus particles of SC-Ad6-PEB1, SC-Ad6-TcdA/B, or PBS.Serum collected at weeks 3, 6, 14, 26, and 36 were titrated to determinebinding endpoint titers for (FIG. 4A) toxin A and (FIG. 4B) toxin Brepresented as geometric mean with standard deviation. (FIG. 4C) Meanneutralizing titers for toxin B were significantly higher in theSC-Ad-TcdA/B than SC-Ad-PEB1 immunized animals (p>0.0079 by MannWhitney). (FIG. 4D) Survival curve for SC-Ad-TcdA/B vaccinated micechallenged with toxin A shows significant protection compared to PBS orPEB1 control animals (p=0.0019 and 0.0021, respectively).

FIGS. 5A-5C. Serum neutralizing antibody responses in immunized SyrianHamsters and protection from lethal spore challenge. Female SyrianHamsters (n=10) were vaccinated intranasally (i.n.) or i.m. with 1×10¹¹virus particles of SC-Ad6-TcdA/B, or i.n. with PBS. Serum collected at6, 12, and 18 weeks after immunization were assayed to determine meanneutralizing titers for (FIG. 5A) toxin A and (FIG. 5B) toxin B(*Adjusted p<0.05 compared to control, **Adjusted p<0.05 compared tocontrol and i.n. route). (FIG. 5C) Survival curve for SC-Ad-TcdA/Bvaccinated animals challenged with UK1 spores shows significantprotection compared to PBS control animals (p<0.0001).

FIGS. 6A-6D. SC-Ad6-TcdA/B provides protection against lethal sporechallenge 45 weeks after single immunization. Female Syrian Hamsters(n=10) were vaccinated i.n. or i.m. with 1×10¹¹ virus particles ofSC-Ad6-TcdA/B, or i.n. with PBS. Serum collected at weeks 6, 12, and 18,25, and 36 weeks after immunization were assayed to determine meanneutralizing titers for (FIG. 6A) toxin A and (FIG. 6B) toxin B(*Adjusted p<0.05 compared to control, **Adjusted p<0.05 compared tocontrol and i.n. route). X-axis break represents the termination of thelow dose challenge study; serum collected at week 36 from remaininganimals in each group (n=5). (FIG. 6C) Survival curve for i.n. (n=5) andi.m. (n=4)SC-Ad-TcdA/B vaccinated animals challenged with UK1 spores 45weeks after single immunization shows significant protection compared toPBS control animals (n=4) (p=0.0027 vs p=0.0067, respectively). (FIG.6D) Week 36 neutralizing toxin A and toxin B neutralizing titers of i.n.immunized animals that survived the challenge compared to non-survivors.

FIG. 7. Blood chemistry of SC-Ad6-TcdA/B vaccinated animals. Groups of10 Syrian hamsters were immunized a single time with 10¹¹ virusparticles of SC-Ad6-TcdA/B by the i.n. or i.m. route. Control animalsreceived i.n. PBS. Blood was collected 3 days after immunization forclinical chemistry.

FIGS. 8A-8C. Blood chemistry and CBC of SC-Ad6-TcdA/B vaccinatedanimals. Groups of 10 Syrian hamsters were immunized a single time with10¹¹ virus particles of SC-Ad6-TcdA/B by the i.n. or i.m. route. Controlanimals received i.n. PBS. Blood was collected ½ of the cohort (n=5 pergroup) (FIG. 8A) 3 and the other ½ of the cohort (n=5 per group) (FIG.8B) 4 days after immunization for clinical chemistry. (FIG. 8C) Bloodwas collected on day 4 from ½ of the hamsters (n=5 per group) for CBC.

FIG. 9. Titration of toxin on Vero cells.

FIG. 10. Titer of Toxin B neutralizing antibodies (nAbs) 26 weeks postchallenge.

FIG. 11. Survival curve 8 weeks after Toxin A challenge.

FIGS. 12A-12B. Schematic representations of exemplary SC-Ad vectors thatcan be used to deliver nucleic acid encoding one or more immunogensaccording to some embodiments. (FIG. 12A) A schematic of an exemplarySC-Ad vector encoding a SARS-CoV-2 Spike variant immunogen and,optionally, one or more adjuvants and/or one more chaff polypeptides.(FIG. 12B) A schematic of an exemplary SC-Ad vector encoding aSARS-CoV-2 Spike variant immunogen and, optionally, one or moreadjuvants and/or one more chaff polypeptides. The polypeptide encodingsequence of pIIIA, which is normally located between 52K and penton, isdeleted.

FIG. 13. SARS-CoV-2 Spike gene and RBD-Sb subdomain genes withrestriction sites.

FIG. 14. Western blot of Spike polypeptide expressed by SC-Ad vector.1°=anti-spike polyclonal (1:1000); 2°=protein A/G-HRP (1:10,000);Substrate=Pico.

FIG. 15. SC-Ad-Spike infects ACE2⁺ cells and forms cell fusion events.

FIG. 16. SC-Ad-Spike infects ACE2⁺ cells, forms cell fusion events, andexpresses adenovirus DNA and adenovirus protein adjuvants.

FIG. 17. Western blot of ACE2 expression in 293-IIIA-ACE2 cells.1°=anti-ACE2 (1:1000); 2°=protein A/G-HRP (1:10,000).

FIG. 18. Western blot of Spike polypeptide expressed by various plasmidexpression vectors.

FIG. 19. Antibody responses in mice 2 weeks after administration ofplasmid vectors expressing Spike polypeptide or negative controlGFP-Luciferase (GL) vector.

FIGS. 20A-20F. IgA and IgG antibody responses in mice 2 (FIGS. 20A, 20C,and 20D) or 6 weeks (FIG. 20B) after intranasal (IN) or intramuscular(IM) administration of SC-Ad expressing Spike polypeptide or negativecontrol SC-Ad expressing Zika protein or buffer (PBS). IFNγ (FIG. 20E)and CD8 T cell counts (FIG. 20F) were also measured.

FIG. 21. Western blot of Spike polypeptide expression byreplication-defective adenovirus (RD-Ad) and SC-Ad expressing SARS-CoV-2spike.

FIG. 22. An exemplary SC-Ad carrying SARS-CoV-2 Spike or RBD withcentralized influenza HA gene H1-CON.

FIG. 23. An exemplary SC-Ad carrying centralized influenza HA geneH1-CON covering H1 hemagglutinins and H1-5 CON covering H1-H5hemagglutinin sequences.

FIG. 24. Serum antibodies generated by plasmid vaccines are increased byco-immunization with granulocyte-macrophage colony-stimulating factor(GM-CSF) adjuvant plasmid.

FIG. 25. Serum antibodies 6 weeks after a single i.n. administration ofSC-Ad HIV Env in combination with SC-Ads expressing genetic adjuvants.

FIG. 26. Vaginal antibodies 6 weeks after a single i.n. administrationof SC-Ad HIV Env in combination with SC-Ads expressing geneticadjuvants.

FIG. 27. Vaginal antibodies after an i.m. administration of SC-Ad HIVEnv in combination with SC-Ads expressing genetic adjuvants and followedby i.m. or i.n. administration of HIV-1 Envelope SOSIP polypeptide.

FIG. 28. A sequence listing for an amino acid sequence (SEQ ID NO:1) ofa SARS-CoV-2 Spike polypeptide.

FIG. 29. A sequence listing for an amino acid sequence (SEQ ID NO:2) ofa SARS-CoV-2 Spike polypeptide lacking an ER retention sequence.

FIG. 30. A sequence listing for an amino acid sequence (SEQ ID NO:3) ofan ectodomain of a SARS-CoV-2 Spike polypeptide.

FIG. 31. A sequence listing for an amino acid sequence (SEQ ID NO:4) ofa receptor binding domain of a SARS-CoV-2 Spike polypeptide.

FIG. 32. A sequence listing for an amino acid sequence (SEQ ID NO:5) ofa receptor binding domain of a SARS-CoV-2 Spike polypeptide fused to anIg polypeptide.

FIG. 33. A sequence listing for an amino acid sequence (SEQ ID NO:6) ofa receptor binding domain of a SARS-CoV-2 Spike polypeptide fused to astreptavidin polypeptide.

FIG. 34. A sequence listing for an amino acid sequence (SEQ ID NO:7) ofa receptor binding domain of a SARS-CoV-2 Spike polypeptide fused to asigma coil polypeptide.

FIG. 35. A sequence listing for an amino acid sequence (SEQ ID NO:8) ofan ectodomain of an ACE2 chaff polypeptide.

FIG. 36. A sequence listing for an amino acid sequence (SEQ ID NO:9) ofan inactivated ectodomain of an ACE2 chaff polypeptide.

FIGS. 37A-37C. Sequence listings for amino acid sequences of adjuvantpolypeptides derived from a C. difficile toxin. (FIG. 37A) A sequencelisting for an amino acid sequence (SEQ ID NO:22) of a TcdA polypeptide.(FIG. 37B) A sequence listing for an amino acid sequence (SEQ ID NO:23)of a TcdB polypeptide. (FIG. 37C) A sequence listing for an amino acidsequence (SEQ ID NO:10) of a TcdA/B fusion polypeptide.

FIG. 38. A sequence listing for an amino acid sequence (SEQ ID NO:11) ofa SARS-CoV-2 ORF1 ab polypeptide.

FIG. 39. A sequence listing for an amino acid sequence (SEQ ID NO:12) ofa SARS-CoV-2 S polypeptide.

FIG. 40. A sequence listing for an amino acid sequence (SEQ ID NO:13) ofa SARS-CoV-2 ORF3 polypeptide.

FIG. 41. A sequence listing for an amino acid sequence (SEQ ID NO:14) ofa SARS-CoV-2 E polypeptide.

FIG. 42. A sequence listing for an amino acid sequence (SEQ ID NO:15) ofa SARS-CoV-2 M polypeptide.

FIG. 43. A sequence listing for an amino acid sequence (SEQ ID NO:16) ofa SARS-CoV-2 ORF3 polypeptide.

FIG. 44. A sequence listing for an amino acid sequence (SEQ ID NO:17) ofa SARS-CoV-2 ORF7 polypeptide.

FIG. 45. A sequence listing for an amino acid sequence (SEQ ID NO:18) ofa SARS-CoV-2 ORF8 polypeptide.

FIG. 46. A sequence listing for an amino acid sequence (SEQ ID NO:19) ofa SARS-CoV-2 N polypeptide.

FIG. 47. A sequence listing for an amino acid sequence (SEQ ID NO:20) ofa centralized H1 influenza hemagglutinin polypeptide.

FIG. 48. A sequence listing for an amino acid sequence (SEQ ID NO:21) ofa centralized H1-5 influenza hemagglutinin polypeptide.

FIG. 49. A sequence listing for a nucleic acid sequence (SEQ ID NO:24)that can encode a TcdA polypeptide derived from a C. difficile toxin.

FIG. 50. A sequence listing for a nucleic acid sequence (SEQ ID NO:25)that can encode a TcdB polypeptide derived from a C. difficile toxin.

FIG. 51. A sequence listing for nucleic acid sequence (SEQ ID NO:26)that can encode a SC-Ad-Spike virus.

FIG. 52. A sequence listing for nucleic acid sequence (SEQ ID NO:27)that can encode a SC-Ad-TcdA/B virus.

FIG. 53. A sequence listing for a SC-Ad6-ΔIII-ΔE3-CMV-Spike-3X-LZLnucleic acid (SEQ ID NO:28).

FIG. 54. A sequence listing for a SC-Ad6-ΔIII-ΔE3-CMV-Spike-PP-3X-LZLnucleic acid (SEQ ID NO:29).

FIG. 55. A sequence listing for a SC-Ad6-ΔIII-ΔE3ADP-I-CMV-Spike-3X-Lnucleic acid (SEQ ID NO:30).

FIG. 56. A sequence listing for a SC-Ad6-ΔIII-ΔE3ADP-I-CMV-Spike-PP-3X-Lnucleic acid (SEQ ID NO:31).

FIG. 57. A sequence listing for a SC-Ad-FZF-657-ΔIIIF-ΔE3-Spike-3X-Lnucleic acid (SEQ ID NO:32).

FIG. 58. A sequence listing for a SC-Ad-FZF-657-ΔIIIF-ΔE3-SpikePP-3X-Lnucleic acid (SEQ ID NO:33).

FIG. 59. A sequence listing for a SC-Ad-FZF-C68-ΔIIIF-ΔE3-Spike-3X-Lnucleic acid (SEQ ID NO:34).

FIG. 60. A sequence listing for a SC-Ad-FZF-C68-ΔIIIF-ΔE3-SpikePP-3X-Lnucleic acid (SEQ ID NO:35).

FIG. 61. A sequence listing for a SC-Ad-F-657-ΔIIIF-ΔE3-Spike-3X-Lnucleic acid (SEQ ID NO:36).

FIG. 62. A sequence listing for a SC-Ad-F-657-ΔIIIF-ΔE3-SpikePP-3X-Lnucleic acid (SEQ ID NO:37).

FIG. 63. A sequence listing for a SC-Ad-F-C68-ΔIIIF-ΔE3-Spike-3X-Lnucleic acid (SEQ ID NO:38).

FIG. 64. A sequence listing for a SC-Ad-F-C68-ΔIIIF-ΔE3-SpikePP-3X-Lnucleic acid (SEQ ID NO:39).

DETAILED DESCRIPTION

This document provides adenovirus vectors and methods and materials forusing adenovirus vectors. In some cases, adenovirus vectors encoding oneor more (e.g., one, two, three, four, five, six, seven, eight, nine,ten, or more) immunogens can be used to deliver immunogens to a mammal(e.g., a human) such that the mammal produces an effective immuneresponse (e.g., an immune response against those immunogens). Forexample, adenovirus vectors encoding one or more immunogens can be usedto deliver immunogens to a mammal (e.g., a human) such that the mammalproduces antibodies against a pathogen, allergen, and/or cancer cellassociated with those immunogens. In some cases, nucleic acid moleculesthat can encode an adenovirus vector encoding one or more immunogens canbe used to deliver immunogens to a mammal (e.g., a human) such that themammal produces an effective immune response (e.g., an immune responseagainst those immunogens). For example, nucleic acid molecules that canencode an adenovirus vector encoding one or more immunogens can be usedto deliver immunogens to a mammal (e.g., a human) such that the mammalproduces antibodies against a pathogen, allergen, and/or cancer cellassociated with those immunogens.

This document also provides adenovirus vectors encoding one or more(e.g., one, two, three, four, five, six, seven, eight, nine, ten, ormore) immunogens (e.g., SC-Ads encoding one or more immunogens), nucleicacid molecules encoding adenovirus vectors encoding one or moreimmunogens, cell lines containing adenovirus vectors encoding one ormore immunogens, methods for using adenovirus vectors encoding one ormore immunogens to deliver the immunogen(s) to cells in vitro or invivo, and methods for using adenovirus vectors encoding one or moreimmunogens to induce immune responses within a mammal (e.g., a human).

An adenovirus vector provided herein (e.g., an adenovirus vectorencoding one or more immunogens) can be derived from any adenovirus. Anadenovirus used to create an adenovirus vector provided herein can beany appropriate serotype (e.g., Ad1-Ad57). In some cases, an adenoviruscan be a replication competent adenovirus. In some cases, an adenoviruscan be a replication defective adenovirus. In some cases, an adenoviruscan be capable of infecting a human cell (e.g., can be a humanadenovirus). In some cases, an adenovirus can be capable of infection anon-human cell (e.g., can be a non-human adenovirus) such as chimpanzeecells. Examples of adenoviruses that can be used to make an adenovirusvector provided herein include, without limitation, Ad5 adenoviruses,Ad6 adenoviruses, ChAdOx1, and ChAdOx2.

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can be a SC-Ad. A SC-Ad can havea genome which lacks all or a portion of at least of one of thefollowing adenovirus nucleic acid sequences:

fiber protein-encoding sequence, V protein-encoding sequence,hexon-encoding sequence, penton base-encoding sequence (also referred toas a pIII-encoding sequence), VA RNA-encoding sequence, pIIIaprotein-encoding sequence (also referred to as a minor capsidprotein-encoding sequence), or other early or late gene product-encodingsequences. Examples of nucleic acid sequences that encode adenoviralpolypeptides include, without limitation, those set forth in GenBank ginumbers 209842, 58478, or 2935210, and/or annotated in GenBank accessionnumbers M73260, X17016, or AF030154. In some cases, a deletion of all ora portion of the nucleic acid encoding one or more of the followingpolypeptides can be engineered into a nucleic acid encoding anadenovirus such that the adenovirus vector does not encode thatfull-length adenovirus polypeptide or a fully functional version of thatadenovirus polypeptide: fiber protein-encoding sequence, Vprotein-encoding sequence, hexon-encoding sequence, penton base-encodingsequence, VA RNA-encoding sequence, pIIIa protein-encoding sequence, orother early or late gene product-encoding sequences. Such deletions canbe any length that results in the deletion of one or more encoded aminoacids and in a reduction or elimination of the normal function of thatpolypeptide. For example, portions of a nucleic acid sequence of anadenovirus can be removed such that the otherwise encoded polypeptidelacks 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more amino acid residues andlacks its normal activity. The portion or portions to be deleted can beremoved from any location along the length of the sequence. For example,a portion of an adenovirus nucleic acid sequence can be removed at the5′ end, the 3′ end, or an internal region of an adenovirus nucleic acidsuch as a fiber protein-encoding sequence, V protein-encoding sequence,hexon-encoding sequence, penton base-encoding sequence, VA RNA-encodingsequence, pIIIa protein-encoding sequence, or other early or late geneproduct-encoding sequences. In some cases, a SC-Ad can be as describedelsewhere (see, e.g., Matchett et al., J. of Virol., 201993(10):e02016-18 (2019); and International PCT Patent ApplicationPublication No. WO 2009/111738).

An adenovirus vector provided herein (e.g., an adenovirus vectorencoding one or more immunogens) can include a nucleic acid sequenceencoding any appropriate immunogen (e.g., a nucleic acid that drivesexpression of any appropriate immunogen). In some cases, an immunogencan be an antigen. An immunogen can be a full-length immunogenicpolypeptide or a portion thereof (e.g., can be derived from animmunogenic polypeptide).

When an immunogenic polypeptide is from a pathogen, the immunogenicpolypeptide can be from any type of pathogen (e.g., a virus, abacterium, a protozoan, a prion, a viroid, or a fungus). In some cases,an immunogenic polypeptide can be a polypeptide expressed by a virus(e.g., a viral polypeptide). For example, an immunogenic polypeptide canbe a polypeptide expressed by a coronavirus (e.g., a beta-coronavirus).Examples of viruses that can express an immunogenic polypeptide include,without limitation, SARS-CoV, HCoV NL63, HKU1, MERS-CoV, SARS-CoV-2,HIV-1, hepatitis B virus, hepatitis C virus, hepatitis D virus,hepatitis E virus, influenza, Ebola virus, Chiningunya virus, Zikavirus, cytomegalovirus, West Nile virus, and those described in Table3-1 of “Learning from SARS: Preparing for the Next Disease Outbreak:Workshop Summary.” Institute of Medicine (US) Forum on MicrobialThreats; Knobler S, Mahmoud A, Lemon S, et al., editors. Washington(DC): National Academies Press (US); 2004. In some cases, an immunogencan be derived from a polypeptide expressed by a bacterium (e.g., abacterial polypeptide). Examples of bacteria that express polypeptidesfrom which an immunogen can be derived include, without limitation,Clostridium (e.g., C. difficile), Staphylococcus aureus (e.g.methicillin-resistant S. aureus), Campylobacter (e.g. Campylobacterjejuni), Mycobacteria (e.g. M. tuberculosis), and Borrelia (B.burgdorferi). Examples of immunogenic polypeptides that can be expressedby a pathogen include, without limitation, C. difficile Toxin A (TcdA)polypeptides, C. difficile Toxin B (TcdB) polypeptides, coronavirusSpike polypeptides, the amino acid sequence set forth in SEQ ID NO:1(see, e.g., FIG. 28), coronavirus nucleoproteins, coronavirus membraneproteins, coronavirus envelope proteins, and coronavirus non-structuralproteins (e.g., coronavirus non-structural proteins 1-16). For example,an immunogenic polypeptide associated with a pathogen can have, or canbe encoded by, a sequence set forth in, for example, National Center forBiotechnology Information (NCBI) Accession Nos: MN938384 and AY772062.

When an immunogenic polypeptide is from an allergen, the immunogenicpolypeptide can be from any type of allergen (e.g., a substance capableof triggering an immune response that results in an allergic reaction).Examples of allergens that can express and/or shed an immunogenicpolypeptide include, without limitation, Fel d 7, Can f1,beta-lactoglobulin, prolamin, parvalbumin, gliadin, Fel dl, chitinase,glutenin, cupin, prolamin, profilins, polcalcins, bet v-1-relatedproteins, 2S albumins, vicilins, legumins, nsLTPs, and Aed a 2. Forexample, adenovirus vectors encoding one or more immunogens describedherein can be used to deliver immunogens to a mammal (e.g., a human)such that the mammal produces antibodies against an allergen associatedwith those immunogens. For example, nucleic acid molecules that canencode an adenovirus vector encoding one or more immunogens can be usedto deliver immunogens to a mammal (e.g., a human) such that the mammalproduces antibodies against an allergen associated with thoseimmunogens. For example, an immunogenic polypeptide associated with anallergen can have, or can be encoded by, a sequence set forth in, forexample, NCBI Accession Nos: NP_001363134.1, AAD56719, NP_001363136.1,NP_001363139.1, P27762.1, P10414.2, P15494.2, P43176.2, NP_001191706.1,NP_001003190.1, XP_030099003.1, or XP_0016577791.

When an immunogenic polypeptide is from a cancer cell (e.g., a cancercell within a mammal having cancer), the immunogenic polypeptide can beexpressed by any cancer cell. For example, an immunogenic polypeptideexpressed by a cancer cell can be a tumor antigen. In some cases, animmunogenic polypeptide expressed by a cancer cell can be a cell surfacetumor antigen. In some cases, an immunogenic polypeptide expressed by acancer cell can be a tumor-associated antigen (TAA; e.g., an antigen,such as an abnormal protein, present on tumor cells). In some cases, animmunogenic polypeptide can be a tumor-specific antigen (TSA; e.g., anantigen present only on tumor cells). Examples of immunogenicpolypeptides that can be expressed by a cancer cell and used asdescribed herein include, without limitation, folate receptor alpha,mucin 1 (MUC-1), human epidermal growth factor receptor 2 (HER-2),estrogen receptor (ER), epidermal growth factor receptor (EGFR), folatereceptor alpha, mesothelin, alphafetoprotein (AFP), carcinoembryonicantigen (CEA), CA-125, epithelial tumor antigen (ETA),melanoma-associated antigen (MAGE), antigens produced by Epstein-BarrViruses, and antigens produced by human papilloma viruses. For example,adenovirus vectors encoding one or more immunogens as described hereincan be used to deliver immunogens to a mammal (e.g., a human) such thatthe mammal produces antibodies against cancer cells associated withthose immunogens. For example, nucleic acid molecules that can encode anadenovirus vector encoding one or more immunogens as described hereincan be used to deliver immunogens to a mammal (e.g., a human) such thatthe mammal produces antibodies against cancer cells associated withthose immunogens. For example, an immunogenic polypeptide associatedwith a cancer cell can have, or can be encoded by, a sequence set forthin, for example, NCBI Accession Nos: XP_002754883.1, AAA03229.1,Q02496.2, AAD33253.1, CEQ32409.1, YP 401631.1, YP 401632.1, orQAR15051.1.

When an immunogenic polypeptide is from a cancer cell (e.g., a cancercell within a mammal having cancer), the immunogenic polypeptide can beexpressed by any type of cancer cell. Examples of such cancers include,without limitation, lung cancers, breast cancers, prostate cancers,liver cancer, kidney cancers, brain cancers, B cell cancers, T cellcancers, ovarian cancers, and skin cancers.

An immunogen can be a full-length immunogenic polypeptide or a portionthereof (e.g., can be derived from an immunogenic polypeptide). Forexample, a nucleic acid sequence encoding an immunogenic polypeptide canbe modified to remove portions of nucleic acid such that the encodedpolypeptide lacks any number of amino acids (e.g., 5, 10, 15, 20, 30amino acids, or all amino acids of the immunogenic polypeptide). In somecases, portions of a nucleic acid sequence encoding an immunogenicpolypeptide can be removed from anywhere along the length of thesequence. For example, portions of the nucleic acid sequence can beremoved at the 5′ end, the 3′ end, or an internal region of the targetnucleic acid. In some cases, an immunogen can be designed to be secretedfrom cells infected with the adenovirus vector encoding the immunogen.For example, a nucleic acid sequence encoding an ER retention sequencecan be removed from a nucleic acid sequence encoding an immunogen (e.g.,such that the encoded immunogen lacks an ER retention sequence). In somecases, an immunogen can be designed to extend from cells infected withthe adenovirus vector encoding the immunogen into the extracellularspace. For example, an immunogen can include an ectodomain of animmunogenic polypeptide. In some cases, an immunogen can bind (e.g., canbe designed to bind) to viral receptor (e.g., an ACE2 polypeptide). Forexample, an immunogen can include a receptor binding domain of animmunogenic polypeptide. Examples of immunogens derived from immunogenicpolypeptides that can be used as described herein include, withoutlimitation, the amino acid sequence set forth in SEQ ID NO:2 (see, e.g.,FIG. 29), the amino acid sequence set forth in SEQ ID NO:3 (see, e.g.,FIG. 30), the amino acid sequence set forth in SEQ ID NO:4 (see, e.g.,FIG. 31), the amino acid sequence set forth in SEQ ID NO:11 (see, e.g.,FIG. 38), the amino acid sequence set forth in SEQ ID NO:12 (see, e.g.,FIG. 39), the amino acid sequence set forth in SEQ ID NO:13 (see, e.g.,FIG. 40), the amino acid sequence set forth in SEQ ID NO:14 (see, e.g.,FIG. 41), the amino acid sequence set forth in SEQ ID NO:15 (see, e.g.,FIG. 42), the amino acid sequence set forth in SEQ ID NO:16 (see, e.g.,FIG. 43), the amino acid sequence set forth in SEQ ID NO:17 (see, e.g.,FIG. 44), the amino acid sequence set forth in SEQ ID NO:18 (see, e.g.,FIG. 45), and the amino acid sequence set forth in SEQ ID NO:19 (see,e.g., FIG. 46).

In some cases, an immunogen described herein can be a variant of awild-type immunogen. For example, a variant of a coronavirus Spikepolypeptide (e.g., a SARS-CoV-2 Spike polypeptide) can comprise orconsist essentially of an amino acid sequence set forth in any one ofSEQ ID NOs:1-4 with one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, or more) amino acid deletions, additions,substitutions, or combinations thereof.

In some cases, an immunogen described herein can have an amino acidsequence with at least 85% sequence identity (e.g., at least 88%sequence identity, at least 90% sequence identity, at least 93% sequenceidentity, at least 95% sequence identity, at least 97% sequenceidentity, at least 98% sequence identity, or at least 99% sequenceidentity) to the amino acid sequence set forth in any one of SEQ IDNOs:1-4.

The percent sequence identity between a particular amino acid sequenceand a sequence referenced by a particular sequence identification numberis determined as follows. First, an amino acid sequence is compared tothe sequence set forth in a particular sequence identification numberusing the BLAST 2 Sequences (Bl2seq) program from the stand-aloneversion of BLASTZ containing BLASTN version 2.0.14 and BLASTP version2.0.14. This stand-alone version of BLASTZ can be obtained online atfr.com/blast or at ncbi.nlm.nih.gov. Instructions explaining how to usethe Bl2seq program can be found in the readme file accompanying BLASTZ.Bl2seq performs a comparison between two sequences using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. Tocompare two nucleic acid sequences, the options are set as follows: -iis set to a file containing the first nucleic acid sequence to becompared (e.g., C:\seql.txt); -j is set to a file containing the secondnucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set toblastn; -o is set to any desired file name (e.g., C:\output.txt); -q isset to -1; -r is set to 2; and all other options are left at theirdefault setting. For example, the following command can be used togenerate an output file containing a comparison between two sequences:C:\B12seq c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r2. To compare two amino acid sequences, the options of Bl2seq are set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (e.g., C:\seql.txt); -j is set to a file containing thesecond amino acid sequence to be compared (e.g., C:\seq2.txt); -p is setto blastp; -o is set to any desired file name (e.g., C:\output.txt); andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two amino acid sequences: C:\B12seq c:\seql.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequencesshare homology, then the designated output file will present thoseregions of homology as aligned sequences. If the two compared sequencesdo not share homology, then the designated output file will not presentaligned sequences. Once aligned, the number of matches is determined bycounting the number of positions where an identical nucleotide or aminoacid residue is presented in both sequences.

A matched position refers to a position in which identical amino acidoccur at the same position in aligned sequences. The percent sequenceidentity is determined by dividing the number of matches by the lengthof the sequence set forth in the identified sequence (e.g., SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4), followed by multiplying theresulting value by 100. For example, an amino acid sequence that has 220matches when aligned with the sequence set forth in SEQ ID NO:2 is 93.2percent identical to the sequence set forth in SEQ ID NO:2 (i.e.,220÷236×100=93.2). It is noted that the percent sequence identity valueis rounded to the nearest tenth. For example, 75.1, 75.2, 75.3, and 75.4is rounded down to 75, while 75.5, 75.6, 75.7, 75.8, and 75.9 is roundedup to 76. It also is noted that the length value will always be aninteger.

In some cases, a coronavirus Spike polypeptide variant can contain theentire amino acid sequence set forth in any one of SEQ ID NOs:1-4,except that the amino acid sequence contains from one to ten (e.g., oneto nine, two to nine, one to eight, two to eight, one to seven, one tosix, one to five, one to four, one to three, two, or one) amino acidadditions, deletions, substitutions, or combinations thereof, providedthat the coronavirus Spike polypeptide variant has the ability to inducean immune response against a coronavirus within a mammal (e.g., ahuman). In some cases, a coronavirus Spike polypeptide variant canconsist essentially of the amino acid sequence set forth in any one ofSEQ ID NOs:1-4 except that the amino acid sequence contains one, two,three, four, or five amino acid residues preceding the articulatedsequence of the sequence identifier (e.g., SEQ ID NO:1), and/or has one,two, three, four, or five amino acid residues following the articulatedsequence of the sequence identifier (e.g., SEQ ID NO:1), provided thatthe coronavirus Spike polypeptide has the ability to induce an immuneresponse against a coronavirus within a mammal (e.g., a human).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can include nucleic acidsequence encoding two or more (e.g., two, three, four, five, six, seven,eight, nine, ten, or more) immunogens from different immunogenicpolypeptides. For example, an adenovirus vector provided herein caninclude nucleic acid sequence encoding an immunogen derived from a firstpathogen (e.g., an immunogen derived from an immunogenic polypeptideexpressed by SARS-CoV-2) and can include nucleic acid sequence encodingan immunogen derived from a second pathogen (e.g., an immunogen derivedfrom an immunogenic polypeptide expressed by a pathogen other thanSARS-CoV-2). In some cases, an adenovirus vector provided herein thatincludes a nucleic acid sequence encoding two or more immunogens derivedfrom an immunogenic polypeptide expressed by different pathogens caninclude nucleic acid sequence encoding a polypeptide comprising,consisting of, or consisting essentially of the amino acid sequence setforth in any one of SEQ ID NOs:1-4 and can include nucleic acid sequenceencoding a polypeptide comprising, consisting of, or consistingessentially of the amino acid sequence set forth in any one of SEQ IDNOs:20-21. When an adenovirus vector provided herein includes nucleicacid sequence encoding two or more immunogens from immunogenicpolypeptides expressed by different pathogens, the adenovirus vector canbe used to induce an immune response against two or more pathogens.

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can include nucleic acidsequence encoding two or more (e.g., two, three, four, five, six, seven,eight, nine, ten, or more) immunogens from the same immunogenicpolypeptide and/or the same pathogen. For example, an adenovirus vectorprovided herein can include nucleic acid sequence encoding two or moreimmunogens derived from the same pathogen. In some cases, an adenovirusvector provided herein that includes a nucleic acid sequence encodingtwo or more immunogens derived from an immunogenic polypeptide expressedby influenza can include nucleic acid sequence encoding a polypeptidecomprising, consisting of, or consisting essentially of the amino acidsequence set forth in SEQ ID NO:20 (see, e.g., FIG. 47) and can includenucleic acid sequence encoding a polypeptide comprising, consisting of,or consisting essentially of the amino acid sequence set forth in SEQ IDNO:21 (see, e.g., FIG. 48).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) that includes a nucleic acidsequence encoding one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, or more) immunogens also can include one ormore regulatory sequences (e.g., an enhancer or a promoter sequence suchas a constitutive, inducible, and/or tissue-specific promoter sequence)to drive transcription of the immunogen(s). Examples of enhancers andpromoters that can be used to drive expression of a nucleic acidsequence encoding one or more immunogens of an adenovirus providedherein include, without limitation, a CMV enhancer sequence, a CMVpromoter sequence, a CAG enhancer sequence, a CAG promoter sequence, aRSV enhancer sequence, a RSV promoter sequence, a Efl alpha enhancersequence, a Efl alpha promoter sequence, a ubiquitin enhancer sequence,a ubiquitin promoter sequence, adenovirus enhancer sequences, andadenovirus promoter sequences. Any appropriate method can be used todetect expression of an immunogen from adenovirus vector infected cells.For example, antibodies that recognize an immunogen can be used todetect the presence or absence of the immunogen.

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can include a nucleic acidsequence encoding one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, or more) adjuvant polypeptides (e.g., nucleicacid that drives expression of one or more adjuvant polypeptides). Forexample, an adenovirus vector can include a nucleic acid sequenceencoding one or more polypeptides that can enhance an immune responsewithin a mammal. In some cases, an adjuvant polypeptide can be acytokine. In some cases, an adjuvant polypeptide can be an immunestimulator. In some cases, an adjuvant polypeptide can be a toxin. Insome cases, an adjuvant polypeptide can accelerate a systemic T cellresponse against a pathogen present within a mammal. In some cases, anadjuvant polypeptide can increase a concentration of antibodies againsta pathogen at a site where the pathogen can enter a mammal's body. Forexample, an adjuvant polypeptide can increase a concentration ofantibodies against a virus (e.g., a coronavirus) at a mucosal site wherethe virus can enter a mammal's body. Examples of adjuvant polypeptidesthat can be encoded by an adenovirus vectors encoding one or moreimmunogens described herein include, without limitation,granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptides,interleukin 4 (IL-4) polypeptides, interleukin 21 (IL-21) polypeptides,CD40 ligand (CD40L) polypeptides, 4-1BB ligand (4-1BBL) polypeptides,transforming growth factor beta (TGF-β) polypeptides, C. difficile toxinpolypeptides (e.g., C. difficile TcdA polypeptides (see, e.g., SEQ IDNO:22), C. difficile TcdB polypeptides (see, e.g., SEQ ID NO:23), and/orthe amino acid sequence set forth in SEQ ID NO:10 (see, e.g., FIG. 37)),and influenza polypeptides (e.g. N polypeptides, H polypeptides, Mpolypeptides, the amino acid sequence set forth in SEQ ID NO:20 (see,e.g., FIG. 47), and/or the amino acid sequence set forth in SEQ ID NO:21(see, e.g., FIG. 48)). In some cases, an adjuvant polypeptide (e.g., SEQID NO:22) can be preceded by an AAT secretory sequence (e.g.,MPSSVSWGILLLAGLCCLVPVSLAEDP; SEQ ID NO:28). In some cases, a nucleicacid sequence encoding an adjuvant polypeptide (e.g., SEQ ID NO:23) canbe preceded by a cleavage site such as a synthetic furin cleavage site(e.g., RGRRSRGRRS; SEQ ID NO:29). Examples of nucleic acid sequencesthat can encoding an adjuvant polypeptide described herein include,without limitation, the nucleic acid sequence set forth in SEQ ID NO:24(see, e.g., FIG. 49) and the nucleic acid sequence set forth in SEQ IDNO:25 (see, e.g., FIG. 50). In some cases, a nucleic acid sequenceencoding an adjuvant polypeptide (e.g., SEQ ID NO:24) can be preceded byan AAT secretory sequence (e.g.,ATGCCTTCATCCGTGTCATGGGGAATCCTGCTGCTGGCTGGACTGTGCTGTCTGGTGCCTGTCTCACTGGCCGAGGACCCT; SEQ ID NO:30). In some cases, a nucleic acidsequence encoding an adjuvant polypeptide (e.g., SEQ ID NO:25) can bepreceded by a cleavage site such as a synthetic furin cleavage site(e.g., AGAGGACGGAGATCAAGAGGAAGGCGCAGC; SEQ ID NO:31). An adjuvantpolypeptide can be a full-length polypeptide or a fragment of anadjuvant polypeptide described herein provided that the fragment has theability to enhance an immune response (e.g., a biologically activefragment). In some cases, an adjuvant polypeptide can be as describedelsewhere (see, e.g., Matchett et al., Vaccines, 8(1):64 (2020)).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can encode (e.g., can bedesigned to encode) a polypeptide that includes an immunogen fused to anadjuvant polypeptide. For example, a nucleic acid sequence encoding animmunogen can be fused to a nucleic acid sequence encoding an adjuvantpolypeptide (e.g., such that the encoded immunogen is fused to theencoded adjuvant polypeptide). An example of an immunogen fused to anadjuvant polypeptide that can be encoded by an adenovirus vectorsencoding one or more immunogens described herein include, withoutlimitation, the amino acid sequence set forth in SEQ ID NO:5 (see, e.g.,FIG. 32).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can include a nucleic acidsequence encoding one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, or more) chaff polypeptides (e.g., nucleic acidthat drives expression of one or more chaff polypeptides). A chaffpolypeptide can be a full-length polypeptide or a fragment thereofprovided that it reduces the rate of entry or inhibits entry of apathogen into a cell within a mammal. In some cases, a chaff polypeptidecan be a soluble polypeptide. For example, a soluble chaff polypeptidecan be a full-length chaff polypeptide or a fragment of a chaffpolypeptide that lacks a transmembrane domain. For example, a solublechaff polypeptide can include an ectodomain of a chaff polypeptide. Insome cases, a chaff polypeptide can target (e.g., target and bind to) aparticular pathogen (e.g., a virus such as a coronavirus) to reduce therate of entry or inhibit entry of the pathogen into a cell within amammal. In some cases, a chaff polypeptide can target (e.g., target andbind to) two, three, four, five, six, or more different pathogens. Insome cases, a chaff polypeptide can include one or more mutations (e.g.,inactivating mutations). Examples of chaff polypeptides that can beencoded by an adenovirus vectors encoding one or more immunogensdescribed herein include, without limitation, full-length ACE2polypeptides and fragments thereof, full-length CD13 polypeptides andfragments thereof, full-length CEACAM1 polypeptides and fragmentsthereof, full-length sialydated polypeptides and fragments thereof,full-length CD46 polypeptides and fragments thereof, full-length nestinpolypeptides and fragments thereof, the amino acid sequence set forth inSEQ ID NO:8 (see, e.g., FIG. 37), and the amino acid sequence set forthin SEQ ID NO:9 (see, e.g., FIG. 38).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can encode (e.g., can bedesigned to encode) a polypeptide that includes an immunogen fused to achaff polypeptide. For example, a nucleic acid sequence encoding animmunogen can include a nucleic acid sequence encoding a chaffpolypeptide (e.g., such that the encoded immunogen is fused to theencoded chaff polypeptide).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can include a nucleic acidsequence encoding one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, or more) marker polypeptides (e.g., nucleicacid that drives expression of one or more marker polypeptides).Examples of marker polypeptides that can be encoded by an adenovirusvector encoding one or more immunogens described herein include, withoutlimitation, fluorescent polypeptides (e.g., GFP, RFP, CFP, and YFP),streptavidin polypeptides, Cre recombinase polypeptides, Caspolypeptides, luciferase polypeptides, betagalactosidase polypeptides,and sodium iodide symporter polypeptides.

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can include a nucleic acidsequence encoding one or more (e.g., one, two, three, four, five, six,seven, eight, nine, ten, or more) polypeptides that can form a multimer(e.g., nucleic acid that drives expression of one or more polypeptidesthat can form a multimer). Examples of polypeptides that can form amultimer that can be encoded by an adenovirus vectors encoding one ormore immunogens described herein include, without limitation,immunoglobulin constant region polypeptides (e.g., an Ig polypeptide),streptavidin polypeptides, and sigma coil polypeptides.

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can encode (e.g., can bedesigned to encode) a polypeptide that includes an immunogen fused to apolypeptide that can form a multimer. For example, a nucleic acidsequence encoding an immunogen can include a nucleic acid sequenceencoding a polypeptide that can form a multimer (e.g., such that theencoded immunogen is fused to the encoded polypeptide that can form amultimer). Examples of immunogens fused to a polypeptide that can form amultimer that can be encoded by an adenovirus vectors encoding one ormore immunogens described herein include, without limitation, the aminoacid sequence set forth in SEQ ID NO:5 (see, e.g., FIG. 32), the aminoacid sequence set forth in SEQ ID NO:6 (see, e.g., FIG. 33), and theamino acid sequence set forth in SEQ ID NO:7 (see, e.g., FIG. 34).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can have a genome that is atleast 85% percent identical (e.g., at least 88% sequence identical, atleast 90% sequence identical, at least 93% sequence identical, at least95% sequence identical, at least 97% sequence identical, at least 98%sequence identical, or at least 99% sequence identical) to a sequenceset forth in any one of SEQ ID NOs:28-39. For example, an adenovirusvector provided herein can have a genome that comprising, consisting of,or consisting essentially of the nucleic acid sequence set forth in SEQID NO:28 (see, e.g., FIG. 53). For example, an adenovirus vectorprovided herein can have a genome that comprising, consisting of, orconsisting essentially of the nucleic acid sequence set forth in SEQ IDNO:29 (see, e.g., FIG. 54). For example, an adenovirus vector providedherein can have a genome that comprising, consisting of, or consistingessentially of the nucleic acid sequence set forth in SEQ ID NO:30 (see,e.g., FIG. 55). For example, an adenovirus vector provided herein canhave a genome that comprising, consisting of, or consisting essentiallyof the nucleic acid sequence set forth in SEQ ID NO:31 (see, e.g., FIG.56). For example, an adenovirus vector provided herein can have a genomethat comprising, consisting of, or consisting essentially of the nucleicacid sequence set forth in SEQ ID NO:32 (see, e.g., FIG. 57). Forexample, an adenovirus vector provided herein can have a genome thatcomprising, consisting of, or consisting essentially of the nucleic acidsequence set forth in SEQ ID NO:33 (see, e.g., FIG. 58). For example, anadenovirus vector provided herein can have a genome that comprising,consisting of, or consisting essentially of the nucleic acid sequenceset forth in SEQ ID NO:34 (see, e.g., FIG. 59). For example, anadenovirus vector provided herein can have a genome that comprising,consisting of, or consisting essentially of the nucleic acid sequenceset forth in SEQ ID NO:35 (see, e.g., FIG. 60). For example, anadenovirus vector provided herein can have a genome that comprising,consisting of, or consisting essentially of the nucleic acid sequenceset forth in SEQ ID NO:36 (see, e.g., FIG. 61). For example, anadenovirus vector provided herein can have a genome that comprising,consisting of, or consisting essentially of the nucleic acid sequenceset forth in SEQ ID NO:37 (see, e.g., FIG. 62). For example, anadenovirus vector provided herein can have a genome that comprising,consisting of, or consisting essentially of the nucleic acid sequenceset forth in SEQ ID NO:38 (see, e.g., FIG. 63). For example, anadenovirus vector provided herein can have a genome that comprising,consisting of, or consisting essentially of the nucleic acid sequenceset forth in SEQ ID NO:39 (see, e.g., FIG. 64).

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) can have a genome that includesone or more of the coding regions set forth in any one of SEQ IDNOs:28-39. For example, a SC-Ad can be designed to have a genome whereall the encoded polypeptides of the SC-Ad have the same amino acidsequence as those polypeptides encoded by the nucleic acid set forth inany one of SEQ ID NOs:28-39.

This document also provides nucleic acid molecules that can encode anadenovirus vector provided herein (e.g., an adenovirus vector encodingone or more immunogens such as a SC-Ad described herein). The term“nucleic acid” as used herein encompasses both RNA and DNA, includingcDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Anucleic acid can be double-stranded or single-stranded. Asingle-stranded nucleic acid can be the sense strand or the antisensestrand. In addition, a nucleic acid can be circular or linear.

This document also provides cells (e.g., cell lines) containingadenovirus vectors described herein (e.g., adenovirus vectors encodingone or more immunogens such as SC-Ads described herein). In cases wherean adenovirus vector lacks all or a portion of at least of oneadenovirus sequences, the cells containing the adenovirus vector canprovide the missing adenovirus polypeptide. For example, when theadenovirus is designed to lack nucleic acid encoding the adenovirusfiber polypeptide, an adenovirus fiber polypeptide-expressing cell linecan be used to generate the adenovirus such that the adenovirus containsthe fiber polypeptide (e.g., the wild-type fiber polypeptide) whilelacking the nucleic acid that encodes the fiber polypeptide (e.g., thewild-type fiber polypeptide). For example, when the adenovirus isdesigned to lack nucleic acid encoding the V polypeptide, an adenovirusV polypeptide-expressing cell line can be used to generate theadenovirus such that the adenovirus contains the V polypeptide (e.g.,the wild-type V polypeptide) while lacking the nucleic acid that encodesthe V polypeptide (e.g., the wild-type V polypeptide). For example, whenthe adenovirus is designed to lack nucleic acid encoding the pIIIapolypeptide, an adenovirus pIIIa polypeptide-expressing cell line can beused to generate the adenovirus such that the adenovirus contains thepIIIa polypeptide (e.g., the wild-type pIIIa polypeptide) while lackingthe nucleic acid that encodes the pIIIa polypeptide (e.g., the wild-typepIIIa polypeptide). In some cases, cells containing adenovirus vectorsdescribed herein can increase the available number of copies of thatvirus by at least 100-fold (e.g., by 100-fold to 15,000-fold, by 500- to10,000-fold, by 5,000- to 10,000-fold, or by 5,000- to 15,000-fold). Avirus can be expanded until a desired concentration is obtained instandard cell culture media (e.g., DMEM or RPMI-1640 supplemented with5-10% fetal bovine serum at 37° C. in 5% CO₂). A viral titer typicallyis assayed by inoculating cells (e.g., A549 or 293 cells) in culture orby quantitating viral genomes by optical density or real-time PCR. Insome cases, cells containing an adenovirus vector provided herein can beused to propagate the adenovirus vector (e.g., to establish a stock ofthe adenovirus vector). For example, a stock of the adenovirus vectorcan be produced by growth in mammalian cells. In some cases, a stock ofthe adenovirus vector can be aliquoted and frozen, and can be stored at−70° C. to −80° C. (e.g., at concentrations higher than thetherapeutically effective dose). In some cases, a stock of theadenovirus vector can be stored in a stabilizing solution. Examples ofstabilizing solutions include, without limitation, sugars (e.g.,trehalose, dextrose, and glucose), amino acids, glycerol, gelatin,monosodium glutamate, Ca′, and Mg′.

In some cases, adenovirus vectors described herein encoding one or moreimmunogens (and/or nucleic acid molecules that can encode an adenovirusvector described herein encoding one or more immunogens) can beformulated into a composition (e.g., a pharmaceutical composition suchas a vaccine composition) for administration to a mammal. For example,adenovirus vectors described herein encoding one or more immunogens(and/or nucleic acid molecules that can encode an adenovirus vectordescribed herein encoding one or more immunogens) can be formulatedtogether with one or more pharmaceutically acceptable carriers(additives), excipients, and/or diluents. Examples of pharmaceuticallyacceptable carriers, excipients, and diluents that can be used in acomposition described herein include, without limitation, sucrose,lactose, starch (e.g., starch glycolate), cellulose, cellulosederivatives (e.g., modified celluloses such as microcrystallinecellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) andcellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol,sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP),polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone(crospovidone), carboxymethyl cellulose,polyethylene-polyoxypropylene-block polymers, and crosslinked sodiumcarboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azodyes, silica gel, fumed silica, talc, magnesium carbonate, vegetablestearin, magnesium stearate, aluminum stearate, stearic acid,antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate,and selenium), citric acid, sodium citrate, parabens (e.g., methylparaben and propyl paraben), petrolatum, dimethyl sulfoxide, mineraloil, serum proteins (e.g., human serum albumin), glycine, sorbic acid,potassium sorbate, water, salts or electrolytes (e.g., saline, protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, and zinc salts), colloidal silica, magnesiumtrisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil.Suitable pharmaceutical formulations depend in part upon the use and theroute of administration. Such forms should not prevent the compositionor formulation from reaching target cells or from exerting its effect.For example, pharmacological compositions injected into the blood streamshould be soluble.

This document also provides methods for using adenovirus vectorsdescribed herein (e.g., adenovirus vectors encoding one or moreimmunogens) and/or nucleic acid molecules that can encode an adenovirusvector described herein. In some cases, adenovirus vectors describedherein and/or nucleic acid molecules that can encode an adenovirusvector described herein can be administered to a mammal (e.g., a human)to increase an immune response (e.g., an increased antibody responseand/or an increased T cell response) against a pathogen (e.g., abacterial or a viral pathogen associated with an immunogen encoded bythe adenovirus vectors). For example, adenovirus vectors describedherein encoding one or more immunogens (and/or nucleic acid moleculesthat can encode an adenovirus vector described herein encoding one ormore immunogens) can be administered to a mammal to provide the mammalwith an immune response effective to reduce the severity of an infectioncaused by a pathogen associated with the immunogen(s) encoded by theadenovirus vectors. In some cases, adenovirus vectors described hereinencoding one or more immunogens (and/or nucleic acid molecules that canencode an adenovirus vector encoding one or more immunogens) can beadministered to a mammal as described herein to provide the mammal withan immune response effective to prevent the mammal from exhibitingsymptoms of an infection caused by a pathogen associated with theimmunogen(s) encoded by the adenovirus vectors. In some cases,adenovirus vectors described herein and/or nucleic acid molecules thatcan encode an adenovirus vector described herein can be administered toa mammal (e.g., a human) to increase a B cell response within themammal. For example, adenovirus vectors described herein encoding one ormore immunogens (and/or nucleic acid molecules that can encode anadenovirus vector described herein encoding one or more immunogens) canbe administered to a mammal as described herein to increase the numberof activated B cells (e.g., plasmablasts, plasma cells, and memory Bcells) within the mammal by, for example, 10, 20, 30, 40, 50, 60, 70,80, 90, 95, or more percent.

In some cases, adenovirus vectors described herein and/or nucleic acidmolecules that can encode an adenovirus vector described herein can beadministered to a mammal (e.g., a human) to increase a number ofantibodies (e.g., antibodies against a pathogen such as a bacterial or aviral pathogen associated with the immunogen(s) encoded by theadenovirus vectors) within the mammal. For example, adenovirus vectorsdescribed herein encoding one or more immunogens (and/or nucleic acidmolecules that can encode an adenovirus vector described herein encodingone or more immunogens) can be administered to a mammal as describedherein to increase the number of antibodies within the mammal by, forexample, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Insome cases, adenovirus vectors described herein and/or nucleic acidmolecules that can encode an adenovirus vector described herein can beadministered to a mammal as described herein to produce antibodiesagainst the immunogen(s) within the mammal for, for example, about oneweek (e.g., from about 1 to about 7 days, from about 1 to about 6 days,from about 1 to about 5 days, from about 1 to about 4 days, from about 1to about 3 days, from about 2 to about 7 days, from about 3 to about 7days, from about 4 to about 7 days, from about 5 to about 7 days, fromabout 2 to about 6 days, from about 3 to about 5 days, from about 2 toabout 4 days, from about 3 to about 5 days, or from about 4 to about 6days).

In some cases, adenovirus vectors described herein and/or nucleic acidmolecules that can encode an adenovirus vector described herein can beadministered to a mammal (e.g., a human) to increase a T cell responsewithin the mammal. For example, adenovirus vectors described hereinencoding one or more immunogens (and/or nucleic acid molecules that canencode an adenovirus vector described herein encoding one or moreimmunogens) can be administered to a mammal as described herein toincrease the number of activated T cells (e.g., cytotoxic T cells andmacrophages) within the mammal by, for example, 10, 20, 30, 40, 50, 60,70, 80, 90, 95, or more percent.

Adenovirus vectors described herein (e.g., adenovirus vectors encodingone or more immunogens) and/or nucleic acid molecules that can encode anadenovirus vector described herein can be administered to anyappropriate mammal (e.g., to increase an immune response against apathogen such as a bacterial or a viral pathogen associated with theimmunogen(s) encoded by the adenovirus vectors within that mammal). Insome cases, the mammal can be a mammal that has not had a previousinfection with a pathogen associated with an immunogen encoded by anadenovirus vector provided herein. In some cases, the mammal can be amammal that has had a previous infection with a pathogen closely related(e.g., genetically related) to a pathogen associated with an immunogenencoded by an adenovirus vector provided herein. In some cases, themammal can be a mammal that has an infection (e.g., an ongoinginfection) with a pathogen associated with an immunogen encoded by anadenovirus vector provided herein. Examples of mammals that can beadministered adenovirus vectors described herein encoding one or moreimmunogens (and/or nucleic acid molecules that can encode an adenovirusvector described herein encoding one or more immunogens) include,without limitation, humans, non-human primates such as monkeys, dogs,cats, horses, cows, pigs, sheep, mice, rats, rabbits, hamsters, bats,raccoons, and ferrets. In some cases, the methods and materialsdescribed herein can be applied to an avian species instead of a mammal.For example, the methods and materials described herein for treating amammal can be applied to chickens and turkeys. In some cases, themethods and materials described herein can be applied to a species ofreptile instead of a mammal. In some cases, the methods and materialsdescribed herein can be applied to a species amphibian of instead of amammal. In some cases, the methods and materials described herein can beapplied to a species of fish instead of a mammal. In some cases, a humancan be administered one or more adenovirus vectors described hereinand/or nucleic acid molecules that can encode an adenovirus vectordescribed herein to increase an immune response against a pathogen(e.g., a bacterial or a viral pathogen associated with an immunogenencoded by the adenovirus vectors).

When administering adenovirus vectors described herein (e.g., adenovirusvectors encoding one or more immunogens) and/or nucleic acid moleculesthat can encode an adenovirus vector described herein (e.g., acomposition such as a vaccine composition including adenovirus vectorsdescribed herein and/or nucleic acid molecules that can encode anadenovirus vector described herein), any appropriate route ofadministration can be used. For example, a composition (e.g., a vaccinecomposition) provided herein can be administered to a mammal (e.g., ahuman) orally (e.g., sublingually) or parenterally (including, withoutlimitation, intranasally, subcutaneously, intramuscularly,intravenously, intradermally, intra-cerebrally, intrathecally, orintraperitoneally). In some cases, the route and/or mode ofadministration of a composition (e.g., a vaccine composition) providedherein can be adjusted for the mammal being treated. In some cases,adenovirus vectors described herein and/or nucleic acid molecules thatcan encode an adenovirus vector described herein can be administered toa mammal via mucosal delivery. In some cases, adenovirus vectorsdescribed herein and/or nucleic acid molecules that can encode anadenovirus vector described herein can be administered to a mammal asdescribed elsewhere (see, e.g., Weaver et al. PLOS ONE, 8(7):e67574(2013)).

Adenovirus vectors described herein (e.g., adenovirus vectors encodingone or more immunogens) and/or nucleic acid molecules that can encode anadenovirus vector described herein (e.g., a composition such as avaccine composition including adenovirus vectors provided herein and/ornucleic acid molecules that can encode an adenovirus vector providedherein) can be administered to a mammal (e.g., a human) in anyappropriate amount (e.g., any appropriate dose). Effective amounts canvary depending on the route of administration, the age and generalhealth condition of the subject, excipient usage, the possibility ofco-usage with other therapeutic treatments such as use of other agents,and the judgment of the treating physician. An effective amount of acomposition containing adenovirus vectors encoding one or moreimmunogens (and/or nucleic acid molecules that can encode an adenovirusvector encoding one or more immunogens) can be any amount that caninduce an immune response in a mammal as described herein withoutproducing significant toxicity to the mammal. For example, an effectiveamount adenovirus vectors encoding one or more immunogens can be, forexample, from about 10⁸ viral particles (vp) to about 10¹⁴ vp (e.g.,from about 10⁸ vp to about 10¹³ vp, from about 10⁸ vp to about 10¹² vp,from about 10⁸ vp to about 10¹¹ vp, from about 10⁸ vp to about 10¹⁰ vp,from about 10⁸ vp to about 10⁹ vp, from about 10⁹ vp to about 10¹⁴ vp,from about 10¹⁰ vp to about 10¹⁴ vp, from about 10¹¹ vp to about 10¹⁴vp, from about 10¹² vp to about 10¹⁴ vp, from about 10¹³ vp to about10¹⁴ vp, from about 10⁹ vp to about 10¹³ vp, from about 10¹⁰ vp to about10¹² vp, from about 10⁹ vp to about 10¹¹ vp, from about 10¹⁰ vp to about10¹² vp, or from about 10¹ vp to about 10¹³ vp). The effective amountcan remain constant or can be adjusted as a sliding scale or variabledose depending on the mammal's response to treatment. Various factorscan influence the actual effective amount used for a particularapplication. For example, the frequency of administration, duration oftreatment, use of multiple treatment agents, and/or route ofadministration may require an increase or decrease in the actualeffective amount administered.

Adenovirus vectors provided herein (e.g., adenovirus vectors encodingone or more immunogens) and/or nucleic acid molecules that can encode anadenovirus vector provided herein (e.g., a composition such as a vaccinecomposition including adenovirus vectors provided herein and/or nucleicacid molecules that can encode an adenovirus vector provided herein) canbe administered to a mammal (e.g., a human) in any appropriatefrequency. The frequency of administration can be any frequency that caninduce an immune response in a mammal without producing significanttoxicity to the mammal. In some cases, adenovirus vectors providedherein and/or nucleic acid molecules that can encode an adenovirusvector provided herein can be administered to a mammal once (e.g., in asingle administration). In some cases, adenovirus vectors providedherein and/or nucleic acid molecules that can encode an adenovirusvector provided herein can be administered to a mammal several times(e.g., as several administrations). For example, the frequency ofadministration can be from about once a day to about every three days,from about once a day to about once a week, from about once a week toabout every 3 weeks, or from about once a week to about every 6 weeks.The frequency of administration can remain constant or can be variableduring the duration of treatment. As with the effective amount, variousfactors can influence the actual frequency of administration used for aparticular application. For example, the effective amount, duration oftreatment, use of multiple treatment agents, and/or route ofadministration may require an increase or decrease in administrationfrequency.

Adenovirus vectors provided herein (e.g., adenovirus vectors encodingone or more immunogens) and/or nucleic acid molecules that can encode anadenovirus vector provided herein (e.g., a composition such as a vaccinecomposition including adenovirus vectors provided herein and/or nucleicacid molecules that can encode an adenovirus vector provided herein) canbe administered to a mammal (e.g., a human) for any appropriateduration. An effective duration for administering or using a compositioncontaining adenovirus vectors encoding one or more immunogens (and/ornucleic acid molecules that can encode an adenovirus vector encoding oneor more immunogens) can be any duration that can induce an immuneresponse in a mammal without producing significant toxicity to themammal. For example, the effective duration can vary from a couple ofdays to one week, from several days to several weeks, or from a few daysto a month. Multiple factors can influence the actual effective durationused for a particular treatment. For example, an effective duration canvary with the frequency of administration, effective amount, use ofmultiple treatment agents, and/or route of administration.

In some cases, an adenovirus vector provided herein (e.g., an adenovirusvector encoding one or more immunogens) and/or a nucleic acid moleculethat can encode an adenovirus vector provided herein (e.g., acomposition such as a vaccine composition including adenovirus vectorsprovided herein and/or nucleic acid molecules that can encode anadenovirus vector provided herein) can be administered to a mammal(e.g., a human) at an effective amount one, two, or three times with oneweek to four weeks between each administration when more than oneadministration is used.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: A Replicating Single-Cycle Adenovirus VaccineAgainst Clostridium difficile

Clostridium difficile causes nearly 500,000 infections and nearly 30,000deaths each year in the U.S. and costs up to $4.8 billion per year. C.difficile infection (CDI) arises from bacteria colonizing the largeintestine and releasing its two toxins, Toxin A (TcdA) and Toxin B(TcdB). This example describes a SC-Ad gene-based vaccine against C.difficile.

Results

Single-Cycle Adenovirus Expressing C. difficile Toxins A and B FusionProtein.

A SC-Ad-TcdA/B vector was generated using adenovirus type 6 (Ad6) (FIG.1). This vector carries a mammalian codon-optimized cDNA that expressesa fusion protein consisting of a secretory leader and the RBDs of TcdAand B separated by two furin cleavage sites. The toxin RBDs were derivedfrom the strain VPI 10463 (toxinotype 0) with glutamine for theasparagine substitutions in putative n-linked glycosylation sites. Thisresulted in a total of eight substitutions in the TcdA RBD and threealterations within the TcdB RBD sequences. Human lung A549 cells wereinfected with SC-Ad6-TcdA/B and cell supernatants and lysates wereanalyzed by western blot using antibodies specific for TcdA and TcdB.The fusion protein was predicted to be 160 kDa with the RBDs of TcdA andTcdB expected to be 100 and 60 kDa, respectively. Under theseconditions, both RBDs and the fusion protein were observed in cells andin concentrated cell supernatants (FIG. 2).

Single Intramuscular Vaccination with SC-Ad6-TcdA/B Induces ImmuneResponses in Mice.

Groups of 10 male and female outbred CD-1 mice were immunized i.m. asingle time with PBS or 10¹⁰ virus particles of SC-Ad6-TcdA/B or anegative control vector SC-Ad6-PEB1 that expresses a mismatched proteinfrom another bacterium, Campylobacter jejuni. Serum was harvested 6weeks after immunization and anti-toxin antibody responses wereevaluated by ELISA and in vitro toxin neutralization assays. Singleimmunization generated significant antibodies against toxin A in themajority of the SC-Ad6-TcdA/B vaccinated animals (FIGS. 3A and 3B).Reciprocal titers were defined as those being significantly higher thanlevels in PBS control mice; therefore, antibody levels are not shown forthe PBS group. Both animal sexes generated antibody responses that weresignificantly higher than in control mice. Female mice had a geometricmean reciprocal toxin A binding titer of 1,467,329 while males hadtiters of 397,964 (FIG. 3A). Female binding titers were significantlyhigher than male titers (p<0.0066 by Mann-Whitney). A similar patternwas observed in the TcdA neutralizing (nAb) titers from the two sexes,with mean titers of 1682 in females and 379 in males (FIG. 3B). However,these differences were not significant (p=0.8004 by Dunn's). When thesewere compared to animals immunized with SC-Ad6-PEB1, those receivingSC-Ad6-TcdA/B had nAb titers that were significantly higher than theirsex matched controls.

Single Intramuscular Vaccination with SC-Ad6-TcdA/B Provides Protectionagainst Toxin Challenge. 8 weeks after single immunization, the micewere challenged with 300 ng (6×LD50) of purified TcdA from List Labs.The recombinant toxin was derived from a Ribotype 087 and Toxinotype 0strain similar to VPI 10463 antigens in the SC-Ad vaccine. Eight out often PBS and PEB1 mice succumbed to the toxin within 24 hours ofchallenge (FIG. 3C). One additional PBS mouse met sacrifice criteria 3days later. Seventeen out of the twenty SC-Ad6-TcdA/B vaccinated micesurvived the challenge. Log-rank comparison of the Kaplan-Meier survivalcurves demonstrated that SC-Ad6-TcdA/B vaccinated animals survivedsignificantly better than PBS or PEB1 control animals. The threeSC-Ad6-TcdA/B vaccinated mice that did not survive had reduced TcdAbinding titers and a complete absence of nAbs (FIGS. 3A and 3B).SC-Ad6-TcdA/B Provides Protection against Toxin Challenge 38 Weeks afterSingle Immunization.

A second set of female CD1 mice were immunized i.m. a single time with10¹⁰ virus particles of SC-Ad6-TcdA/B, SC-Ad6-PEB1, or PBS (n=5 pergroup). This single vaccination of SC-Ad6-TcdA/B generated strongantibody responses with reciprocal endpoint binding titers for TcdA andTcdB that climbed above 100,000 over 26 weeks (FIGS. 4A and 4B). At week26, the average reciprocal TcdB nAb titer in the SC-Ad-TcdA/B groupreached 174 (FIG. 4C). At week 38, the mice were challenged with 300 ng(6×LD50) of TcdA. All PBS and control SC-Ad-PEB1 vaccinated animalssuccumbed to the toxin within 48 hours (FIG. 4D). In contrast, allanimals in the SC-Ad C. difficile vaccine group survived.

Pilot Toxicology and Efficacy Testing in Hamsters.

Groups of 10 Syrian hamsters were immunized a single time with 10¹¹virus particles of SC-Ad6-TcdA/B by the i.n. or i.m. route. Controlanimals received i.n. PBS. Blood was collected 3 days after immunizationfor clinical chemistry. These revealed no significant differences inblood chemistry (FIG. 7).

Single Intranasal or Intramuscular Vaccination with SC-Ad6-TcdA/BInduces Immune Responses in Hamsters.

Serum was collected from the hamsters 6, 12, and 18 weeks afterimmunization and anti-toxin antibody responses were evaluated. Singleimmunization with SC-Ad6-TcdA/B generated significant serum nAbs againsttoxin A regardless of route. These antibody levels increased over thecourse of the study (FIG. 5A). While i.m. immunization produced highermean nAbs against toxin A than the i.n. group, these were notsignificantly different until week 18 (p=0.0336 by Dunn's). Toxin Bantibodies were detectable by ELISA at week 6. However, significantlevels of nAbs against toxin B took longer to develop (FIG. 5B). Alli.m. immunized animals and 6/10 of the i.n. immunized animals hadsignificant toxin B nAb levels by week 12. At week 18, all i.m. and i.n.immunized animals had significant toxin B nAbs with mean reciprocaltiters of 2084 and 229, respectively.

Single Intramuscular Vaccination with SC-Ad6-TcdA/B Provides Protectionagainst C. difficile Spore Challenge in Hamsters.

CDI can be induced in Syrian hamsters when sensitized with clindamycin.This model mimics the fecal-oral route of transmission by deliveringpurified C. difficile spores orogastrically producing symptoms similarto those observed in patients with CDI. Various toxin isoforms have beenidentified within clinical isolates of C. difficile. Recent studiesreport that the BI/NAP1/027 strain of C. difficile is the most prevalentcause of CDI in North America. Given its clinical relevance andexpression of heterologous toxin isoforms, vaccine efficacy was testedusing spores from the UK1 (BI/NAP1/027) strain. Hamsters wereadministered clindamycin intraperitoneally 24 hours before receiving10,000 spores 20-21 weeks after a single immunization. All of the PBSimmunized animals succumbed to the spore challenge (FIG. 5C).Strikingly, all of the SC-Ad6-TcdA/B vaccinated hamsters survived to theend of the study regardless of vaccine administration route. Log-rankcomparison of the Kaplan-Meier survival curves demonstrated that bothi.n. and i.m. SC-Ad6-TcdA/B vaccinated animals had significantly bettersurvival than PBS control animals. Weight loss was observed in allanimals over the course of the experiment, but weight loss inSC-Ad6-TcdA/B animals either stabilized or began to reverse by day 7.

SC-Ad6-TcdA/B Provides Protection against Lethal Spore Challenge 45Weeks after Single Immunization.

A second group of 10 female Syrian hamsters were immunized a single timewith 10¹¹ virus particles of SC-Ad6-TcdA/B either i.n. or i.m. or withPBS. Blood was collected 3 or 4 days after immunization and tested usingthe same analyte panel as before. Similarly, no significant differenceswere observed between vaccinated and unvaccinated on days 3 or 4 (FIGS.8A and 8B). At day 4, CBCs were measured in half of the animals. Therewere significant increases in the percentage of neutrophils and acorresponding decrease in the percentage of lymphocytes in animalsreceiving vaccine compared to controls; however, comparison of thenumber of neutrophil and lymphocyte showed no differences (FIG. 8C).I.m. immunized animals saw increases in their platelets andplateletcrit, although these were still within normal ranges.

Serum collected at weeks 6, 12, and 18 showed increasing toxin A and BnAbs as in the first vaccination study (FIGS. 6A and 6B). At week 24,half of the hamsters in each group were sensitized with clindamycingiven orogastrically (30 mg/kg) and then were challenged with 200 sporesof C. difficile 5 days later. This low dose challenge surprisinglyinduced no symptoms or indications of C. difficile infection in anyhamsters including the PBS controls. Serum antibodies collected at thetermination of this challenge revealed no increases in toxin A or Bantibodies due to the pathogen challenge compared to unchallengedanimals in the cohort. The unchallenged animals were followed for anadditional 20 weeks. In this period, one PBS and one i.m. immunizedanimals became moribund and had to be euthanized at week 42 and 44,respectively.

The remaining animals were challenged at week 45 using the high dose10,000 spore challenge. All PBS immunized animals met endpoint criteriaand were euthanized (FIG. 6C). Two intranasally-immunized animals alsosuccumbed to the challenge. All animals in the i.m. vaccine groupsurvived spore challenge. Log-rank comparison of these survival curvesshowed significant differences in the survival of both i.n. and i.m.SC-Ad6-TcdA/B vaccinated animals compared to PBS. Toxin nAbs levelsbefore challenge (week 36), correlated with survival in the groups (FIG.6D). Animals in the i.n. group that survived spore challenge had hightoxin nAbs prior to challenge. In contrast, animals in this group thatdid not survive had lower toxin nAbs before they were challenged.Protection against C. difficile challenge was observed 10 months afteronly a single immunization with SC-Ad vaccine.

Materials and Methods Cell Culture

A549 cells and Vero cells were purchased from the American Type CultureCollection. The 293-IIIA cells were generated as described elsewhere(Crosby et al., Virology 462-463:158-165 (2014)). All cells weremaintained at 37° C. in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% heat inactivated fetal bovine serum (HI-FBS;HyClone) and penicillin/streptomycin at 100 U/mL (Invitrogen).

Single-Cycle Adenovirus Expressing C. difficile TcdA/B Fusion

A codon-optimized cDNA encoding a novel fusion of the receptor bindingdomains of C. difficile toxins A and B was synthesized by Genscript.This cDNA contains the secretory leader sequence from alpha-1antitrypsin (AAT) to facilitate secretion of the fusion protein. Thereceptor binding domains are separated by two furin cleavage sites toliberate the two Tcds from each other during secretion from mammaliancells. This cDNA was inserted into the shuttle plasmids pAd6-NdePfl andwas recombined into SC-Ad6 as described elsewhere (Crosby et al.,Virology 462-463:158-165 (2014), Crosby et al., J. Virol.91(2):e00720-16 (2017); and Crosby et al., J. Virol. 89:669-675 (2015))to generate SC-Ad6-C. diff. Control SC-Ad6 viruses expressingGFP-Luciferase or Campylobacter jejuni PEB1 were also used. Viruses wererescued, amplified, and purified as described elsewhere (Crosby et al.,Virology 462-463:158-165 (2014), Crosby et al., J. Virol.91(2):e00720-16 (2017); and Crosby et al., J. Virol. 89:669-675 (2015)).Virus comparisons were based on virus particles.

Western Blotting

A549 cells were infected with SC-Ad6-TcdA/B or SC-Ad-GL, which expressesGFP-Luciferase, with 10⁴ virus particles/cell. 24 hours after infection,the media was replaced with serum-free DMEM. 24 hours later, this mediawas collected and concentrated using Amicon Ultra-15 30 k (Millipore).The cells were harvested using Triton-X lysis buffer supplemented withComplete™ Protease Inhibitor Cocktail (Roche). Media concentrates andcell lysates were analyzed by western blot using antibodies against C.difficile toxin A or B (1:1000; List Biological Labs, Inc.) followed bya goat anti-chicken horseradish peroxidase secondary antibody (1:1000;Invitrogen). SuperSignal West Dura (Thermo Scientific) was added andblots were imaged on an In Vivo F Station (KODAK).

Animals

Male and female outbred CD-1 mice (Charles River Laboratories) andfemale Golden Syrian hamsters (Envigo) were housed in the Mayo ClinicAnimal Facility. All animal handling and experiments were carried outaccording to the provisions of the Animal Welfare Act, PHS AnimalWelfare Policy, the principles of the NIH Guide for the Care and Use ofLaboratory Animals, and the policies and procedures of the InstitutionalAnimal Care and Use Committee at Mayo Clinic.

Immunizations and Sample Collection

Mice were anesthetized with isoflurane and immunized by theintramuscular (i.m.) route with 10¹⁰ vp of the indicated vaccine.Hamsters were anesthetized with isoflurane and immunized intramuscularly(i.m.) or intranasally (i.n.) routes with 10¹¹ vp of the vaccine. Inmice, serum was collected from the facial vein at the indicated timepoints. At various time points, hamsters were anesthetized and blood wascollected from the jugular vein.

Enzyme-Linked Immunosorbent Assay (ELISA)

Immulon 4 HBX plates (Thermo) were coated with 100 ng/well of either C.difficile A or B toxoids (List Biological Labs, Inc.) in 1×phosphate-buffered saline (PBS) overnight. Wells were washed and blockedwith 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at roomtemperature (RT) for 2 hours. After washing with TBST, half logdilutions of each serum sample were plated in triplicate and incubatedfor 3 hours at RT. Wells were washed and 100 μL of 1:10,000 goatanti-mouse IgG-horseradish peroxidase (Thermo Fisher Scientific Inc.)was added to each well. Plates were incubated for 2 hours at RT. Wellswere washed and 50 μL of 1 step Ultra TMB ELISA (Thermo FisherScientific Inc.) were added to each well. When color developed, 50 μL of2M H2504 were added. OD450 was determined with a BioTek Synergy H1Hybrid Multi-Mode Reader. Reciprocal titers were statistically definedbased on 95% confidence interval.

Cytotoxicity and Neutralization Assays

Cytotoxicity of C. difficile toxin A and toxin B were determined on Verocells using the method described elsewhere (Donald et al., Microbiology159:1254-1266 (2013)). Vero cells were used in place of IMR-90 as theyhave similar sensitivity to Toxin B compared to IMR-90 cells, but havean increased sensitivity to toxin A. Vero cells were plated at 10⁴ cellsper well in a 96 well plate. C. difficile toxin A or toxin B (ListBiological Laboratories, Inc) was serially diluted in DMEM supplementedwith 10% HI-FBS and added to cells 24 hours after plating. Three dayslater, cell viability was determined using the bioluminescentCellTiter-Glo reagent (Promega). An EC50 was determined to be equivalentto the amount of toxin causing 50% reduction in luminescence by fittingthe data with a four-parameter equation. Toxin neutralization wasdetermined using serial dilutions of mouse or hamster serum mixed witheight times the EC50 value determined in the cytotoxicity assay. Themixtures were incubated at 37° C. for 90 minutes in a humidifiedincubator (5% CO₂) before being added to Vero cells on 96 well-plates.Three days later, cell viability was determined with Celltiter-Glo. Afour-parameter regression response was fitted to the luciferase relativelight units (RLU) values derived from the serum dilutions. Neutralizingantibody (nAb) titers were expressed as the derived sample dilution thatexhibited a 50% reduction in cytotoxicity. If a serum titration failedto generate 50% inhibition within the range of concentrations tested, atiter value of ½ of the highest serum concentration tested was ascribedto it.

Challenge with Recombinant C. difficile Toxin A in Mice

Immunized mice were challenged intraperitoneally (i.p.) with 300 ng ofC. difficile toxin A (List Biological Laboratories, Inc). Followingtoxin challenge, mice were monitored every 3 hours for the first 30hours, followed by monitoring at 6-hour intervals for 72 hours, and thenevery 12 hours from day 3 through day 7 (168 hours). Mice were monitoredfor clinical signs. Briefly, animals' symptoms were scored as normal,lethargic, abnormal or moribund. Moribund animals were euthanizedimmediately and recorded. The survival rate was determined for eachtreatment group.

Hematology and Clinical Chemistry in Hamsters

Blood was collected for clinical chemistry analysis (200 μL into lithiumheparin tubes; Greiner Bio-One) and for complete blood count (CBC, 100μl in K2 EDTA tubes; Greiner Bio-One). Blood chemistry was analyzed witha Piccolo Xpress Analyzer (Abaxis) and CBCs were determined with VetScanHM5 hematology analyzer (Abaxis). Analyte parameters for the two testsare shown in Table 1 and Table 2.

TABLE 1 Veterinary Hematology Parameters Measured on the Abaxis VetScanHM5 Analyzer Results Table. Parameter Abbreviation Units Sodium NA+mmol/L Potassium K+ mmol/L Total Carbon Dioxide tCO2 mmol/L Chloride CL−mmol/L Glucose GLU mg/dL Calcium CA mg/dL Blood Urea Nitrogen BUN mg/dLCreatinine CRE mg/dL Alkaline Phosphatase ALP U/L AlanineAminotransferase ALT U/L Aspartate Aminotransferase AST U/L TotalBilirubin TBIL mg/dL Albumin ALB g/dL Total Protein TP g/dL

TABLE 2 Blood Chemistry Parameters Measured on the Piccolo XpressChemistry Analyzer. Parameter Abbreviation Units Total White Blood Cellcount WBC 10{circumflex over ( )}9L Lymphocyte count LYM 10{circumflexover ( )}9L Monocyte count MON 10{circumflex over ( )}9L Neutrophilcount NEU 10{circumflex over ( )}9L Eosinophil count EOS 10{circumflexover ( )}9L Basophil count BAS 10{circumflex over ( )}9L Lymphocytepercentage LYM% % Monocyte percentage MON% % Neutrophil percentage NEU%% Eosinophil percentage EOS% % Basophil percentage BAS% % Red Blood Cellcount RBC 10{circumflex over ( )}12L Hemoglobin HGB g/dL Hematocritpercentage HCT % Mean Corpuscular Volume MCV fL Mean CorpuscularHemoglobin MCH pg Mean Corpuscular Hemoglobin Concentration MCHC g/dlRed Cell Distribution Width, coefficient of RDWc % variation % Red CellDistribution Width RDWs fL Platelet count PLT 10{circumflex over ( )}9LMean Platelet Volume MPV fL Platelet crit % PCT % Platelet DistributionWidth, coefficient of PDWc % variation % Platelet Distribution WidthPDWs fLChallenge with C. difficile Spores in Hamsters

Prior to challenge, hamsters were housed individually in ventilatedcages. In the low dose challenge, hamsters were sensitized for infectionusing a clindamycin phosphate (Sigma-Aldrich) antibiotic solution (30mg/kg of body weight) delivered orogastrically via a feeding needle.Five days later, the hamsters were challenged orogastrically with 200spores from C. difficle strain UK1. Since low dose spore challenge didnot induce symptoms in hamsters in our hands, a high dose challenge withmodified clindamycin administration was used. In this challenge,hamsters were sensitized with clindamycin phosphate antibiotic solution(10 mg/kg of body weight) by the intraperitoneal route rather than theorogastric route. The hamsters were then challenged 24 hours laterorogastrically with 10⁴ UK1 spores. In both the high and low dosechallenge, the hamsters were monitored 4 times per day followinginfection by assessing them individually in a microbiological safetycabinet for several parameters, including presence and severity of wettail, loose feces, diarrhea, weight loss, activity level, starey coat,sunken eyes, hunched posture and response to stimulus. A scoring system,based on severity of changes observed (ranging from 0-3 for eachparameter), was used to quantify the condition of the animals. Animalswere euthanized and considered to have succumbed to disease when theyeither reached a score were moribund, or suffered weight loss in excessof 20%.

Statistical Analysis

Prism 8 Graphical software was used for all statistical analyses.

Example 2: Single-Cycle Adenovirus Vectors Expressing a SARS-CoV-2Polypeptide

A full-length codon-optimized SARS-CoV-2 Spike cDNA (FIG. 13) or RBD-Sbsubdomains were inserted into pAd6-ΔIII-ΔE3 with and without geneticadjuvant or chaff genes (FIG. 12) and rescued in 293-IIIA cells.CsCl-purified SC-Ad6-Spike produced monomer, dimer, and trimers of spikeas well as cleaves S2 domain after infection of A549 human lung cells asdemonstrated by western blot with anti-spike antibody (FIG. 14).SC-Ad6-Spike induced cell-cell fusion and syncytia in cells engineeredto express its receptor ACE2 (FIG. 15). When these syncytia wereexamined, they contained abundant amounts of adenovirus proteins asdemonstrated by immunohistochemical staining for adenovirus hexon withAdenoX Rapid Titer reagent (FIG. 16). When SC-Ad6-Spike was used toimmunize BALB/c mice by the intranasal (i.n.) or intramuscular (i.m.)route, the virus induced strong spike antibodies within 2 weeks asdemonstrated by ELISA using 1/1000 dilutions of mouse sera (FIG. 20A).These were class-switched IgG antibodies and they were significantlyhigher than negative control mice immunized with PBS buffer or SC-Adexpressing Zika E (p<0.0001 by one-way ANOVA). At 6 weeks afterimmunization, IgG antibodies remained elevated in sera. Notably,intranasal immunization also generated IgA antibodies indicative ofresponses at mucosal barriers (FIG. 20B). Dose-finding studies in BALB/cmice demonstrated significant IgG antibodies responses were generated 2weeks after a single i.n. or i.m. immunization with 10⁸ viral particlesof SC-Ad-Spike (** p<0.01, **** p<0.0001 by ANOVA, FIG. 20C).

A replication-defective Ad6 (RD-Ad6) with an E1 deletion was constructedwith the same Spike expression cassette. RD-Ad6-Spike and SC-Ad-Spikewere used to infect A549 human lung cells with different numbers ofvirus particles (vp) per cell. When cell lystates were examined 24 hourslater by western blot for the Spike protein, this revealed that SC-Adexpressed high levels of Spike protein when 10² or 10⁴ vp of the virus(FIG. 21). In contrast, RD-Ad-Spike produce no detectable Spike proteinwith 10² particles of virus and only low levels of Spike protein with10⁴ vp of virus.

SC-Ad expressing coronavirus Spike or RBD proteins can be modified bythe addition of influenza genes to generate a combined coronavirus andinfluenza virus vaccine. For example, SC-Ad6 containing Spike and acentralized H1 consensus influenza hemagglutinin (H1-CON) gene or SC-Ad6containing the Spike RBD domain and a centralized H1 consensus influenzahemagglutinin (H1-CON) gene (FIG. 22). Alternatively, an SC-Adexpressing Spike or RBD could be co-immunized with an SC-Ad expressingtwo influenza consensus immunogens. For example, SC-Ad6 containingcentralized H1 consensus influenza H1-CON and H1-5 centralized HA geneH1-5-CON (FIG. 23).

Example 3: Single-Cycle Adenovirus Vectors Expressing Genetic Adjuvants

10⁹ viral particles (vp) of SC-Ad6 expressing clade C gp140 fromSHIV-1157ipd3N4 was used to immunize BALB/c mice by the i.n. route incombination with 10⁹ SC-Ads expressing 4-1BBL, GMCSF, C. diff toxinfragment TcdA/B or a non-specific adenovirus control expressingGFP-Luciferase. ELISAs using serum collected 6 weeks after singleimmunization demonstrated significant increases in antibody isotypes byGMCSF and TcdA/B (p<0.05 or less for all IgGs) (FIG. 25). When vaginalwashes were assayed for IgA at the same time point, this revealedsimilar trends with highest mucosal IgA mediated by i.n. co-delivery ofTcdA/B adjuvant (FIG. 26).

SC-Ad-GMCSF and TcdA/B were tested again by the i.m. route with 10-foldmore SC-Ad. SC-Ad-IL-21 adjuvant was also added for its ability tostimulate Tfh and other T cells. In this case, i.m. injections wereadministered to the quadriceps muscles near to the vaginal sample site.6 weeks after this single higher dose i.m. immunization, ELISA with1/2000 dilutions of sera showed increased env IgG levels by SC-Ad-GMCSF,TcdA/B and IL-21 (p<0.05 vs PBS). SC-Ad-IL-21 provided even higherantibody levels than GMCSF or TcdA/B (p<0.0001 vs PBS). When vaginalwash samples were tested for IgG, all SC-Ad-1157 animals had increases,but only SC-Ad-IL-21 adjuvant reached significance (p<0.05 vs PBS). Whenvaginal washes were assayed for IgA at the same time point, thisrevealed similar trends with higher mucosal IgA in most animals in theGMCSF, TcdA/B, and IL-21 groups, only the IL-21 group reached p<0.05).Soluble HIV SOSIP envelope protein was used to boost the responsesgenerated by SC-Ad. Each of the i.m. SC-Ad-1157+SC-Ad adjuvant immunizedmice was boosted with 5 μg of clade C CZA97 SOSIP.v4.2-M6.IT mixed withthe NKT cell adjuvant alphaGalCer. One half of the mice were boosted bythe i.m. route and one half were boosted by the i.n. route. 2 weekslater, vaginal washes were collected and assayed for IgA or IgGantibodies against clade C env (FIG. 27). These data showed a strongbias in antibody responses based on the route of delivery of the SOSIPprotein boost. i.m. SOSIP increased vaginal IgG levels generated by i.m.SC-Ad-1157 and SC-Ad GFP-Luc or GMCSF better than i.n. protein. Incontrast, i.n. SOSIP protein boost strongly amplified vaginal IgA levelsin mice that were primed by the i.m. route with SC-Ad-1157 with thestrongest SC-Ad adjuvants: GMCSF, TcdA/B, and IL-21. TheSC-Ad-1157+SC-Ad-GFP-Luc group showed robust IgG responses when primedand boosted intramuscularly, but failed to generate a strong IgGresponse when the SOSIP was given i.n. Furthermore, either of thesecombinations failed to generate IgA responses. This would suggest thatgenetic adjuvants that are given in place of SC-Ad-GFP-Luc prime theanimals to drive the IgA responses we observe when they are boosted i.n.also show that priming at mucosal surfaces. The protein administered tounprimed animals generated little IgG or IgA response in vaginal washes.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A single-cycle adenovirus (SC-Ad), wherein saidSC-Ad comprises a genome lacking at least a portion of a nucleic acidsequence that encodes an adenovirus polypeptide, wherein said SC-Adcomprises said adenovirus polypeptide, and wherein said SC-Ad comprisesa nucleic acid sequence encoding a TcdA/B fusion polypeptide.
 2. TheSC-Ad of claim 1, wherein said adenovirus polypeptide is selected fromthe group consisting of a fiber polypeptide, a V polypeptide, a hexonpolypeptide, a penton base polypeptide, and a pIIIa polypeptide.
 3. TheSC-Ad of claim 1, wherein said TcdA/B fusion polypeptide comprises theamino acid sequence set forth in SEQ ID NO:10 or comprises an amino acidsequence at least 85 percent identical to the sequence set forth in SEQID NO:10.
 4. The SC-Ad of claim 1, wherein said TcdA/B fusionpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:10.5. A composition comprising a single-cycle adenovirus (SC-Ad), whereinsaid SC-Ad comprises a genome lacking at least a portion of a nucleicacid sequence that encodes an adenovirus polypeptide, wherein said SC-Adcomprises said adenovirus polypeptide, and wherein said SC-Ad comprisesa nucleic acid sequence encoding a TcdA/B fusion polypeptide.
 6. Thecomposition of claim 5, wherein said adenovirus polypeptide is selectedfrom the group consisting of a fiber polypeptide, a V polypeptide, ahexon polypeptide, a penton base polypeptide, and a pIIIa polypeptide.7. The composition of claim 5, wherein said TcdA/B fusion polypeptidecomprises the amino acid sequence set forth in SEQ ID NO:10 or comprisesan amino acid sequence at least 85 percent identical to the sequence setforth in SEQ ID NO:10.
 8. The composition of claim 5, wherein saidTcdA/B fusion polypeptide comprises the amino acid sequence set forth inSEQ ID NO:10.
 9. A method for inducing, in a mammal, an immune responseagainst C. difficile, wherein said SC-Ad comprises a genome lacking atleast a portion of a nucleic acid sequence that encodes an adenoviruspolypeptide, wherein said SC-Ad comprises said adenovirus polypeptide,wherein said SC-Ad comprises a nucleic acid sequence encoding a TcdA/Bfusion polypeptide, wherein said method comprises administering saidSC-Ad to said mammal under conditions wherein said SC-Ad infects a cellof said mammal, and wherein expression of said TcdA/B fusion polypeptidein said cell leads to induction of said immune response.
 10. The methodof claim 9, wherein said mammal is a human.
 11. The method of claim 9,wherein said administering comprises an intramuscular administration ofsaid SC-Ad.
 12. The method of claim 9, wherein said adenoviruspolypeptide is selected from the group consisting of a fiberpolypeptide, a V polypeptide, a hexon polypeptide, a penton basepolypeptide, and a pIIIa polypeptide.
 13. The method of claim 9, whereinsaid TcdA/B fusion polypeptide comprises the amino acid sequence setforth in SEQ ID NO:10 or comprises an amino acid sequence at least 85percent identical to the sequence set forth in SEQ ID NO:10.
 14. Themethod of claim 9, wherein said TcdA/B fusion polypeptide comprises theamino acid sequence set forth in SEQ ID NO:10.